JP2002516097A - Human calmodulin-related protein - Google Patents

Abstract

(57) Abstract The present invention provides a human nucleic acid methylase (HNAM) and a polynucleotide identifying and encoding the same. The present invention also provides expression vectors and host cells, antibodies, agonists and antagonists. Furthermore, the present invention provides a method for diagnosing, treating, or preventing a disease associated with HNAM expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid and amino acid sequences of the human calmodulin-related proteins, and diagnosis of cell proliferative disorders and neurological diseases, treatment, and to a use of these sequences in the prevention.

[0002]Background of the Invention) In almost all eukaryotic cells, calcium (Ca²⁺), Cell proliferation, neurotransmission
Various cellular processes, including secretion of substances, glycogen metabolism, and skeletal muscle contraction
Functions as an intracellular signaling molecule. In stationary phase cells,
Ca in itsol²⁺Is very low (<10^-7M) However
Cells are stimulated by external signals such as nerve impulses or growth factors
And Ca in the cytosol²⁺Increases approximately 50-fold. Ca²⁺The inflow of
Plasma membrane Ca²⁺Channels open and Ca from intracellular stores such as the endoplasmic reticulum ²⁺ Is released. Ca²⁺Is, for example, a protein kinase C
Such regulatory enzymes are directly activated, which initiates signal transduction pathways. Ma
The Ca²⁺Is a specific Ca such as calmodulin²⁺Also binds to binding proteins,
The protein is targeted at various target proteins such as enzymes, membrane transport pumps, and ion channels.
Activates the protein.

[0003] Many Ca ²⁺ binding proteins have been characterized by the presence of one or more EF-hand Ca ²⁺ binding motifs (Reviewed in Cello, MR et al. (1996) Gui.
debook to the Calcium-Binding Proteins, Oxford University Press, New Yor
k, NY, pp. 15-20, 34-43, and 152-155.). The EF hand motif consists of 12 amino acids flanking the α helix. Usually 1, 3, 5, 7, 9, and 1
An acidic residue is conserved in the second position and glycine is often conserved in the sixth position. At least 250 EF hand proteins belonging to 39 subfamilies have been identified in various tissues. For example, calmodulin
It is a widely conserved EF hand protein that regulates cell growth and cell cycle progression. Tropnin C is a muscle-specific calmodulin-like protein involved in actin-induced muscle contraction. S100β is an EF hand protein that is highly expressed in the nervous system, especially in glial cells. Recently, a messenger RNA (m) encoding a calmodulin-related protein called NB-1
RNA) was isolated from normal human breast epithelial cells (HMECs) (Yaswen,
(1990) Proc. Natl. Acad. Sci. USA 87: 7360-7364.). NB-1 has four EF hands and shares 85% sequence identity with calmodulin at the amino acid level. NB-1 mRNA expression is reduced by a factor of less than 50 in HMECs that have been transformed into tumors in vitro. Also, NB-1 transcripts are detected in normal breast, prostate, cervical, and epithelial tissues, but not in immortalized cell lines derived from tumors of these tissues. These observations suggest that NB-1 expression is associated with tumor suppression.

[0004] Abnormal expression of the EF hand Ca ²⁺ binding protein has been linked to the progression and diagnosis of certain disease states (Celio, supra). For example, in tumor cells, the concentration of calmodulin is several times higher than that of NB-1. Similarly, S100β
Is elevated in the cerebrospinal fluid and blood of patients suffering from neurological disorders due to cerebral infarction, transient ischemic attacks, hemorrhage, head trauma, and Down's syndrome. Furthermore,
S100β and other neuron-specific EF hand proteins can prevent neurodevelopmental disorders such as, for example, Alzheimer's disease, Parkinson's disease, and Huntington's disease.
In addition, antibodies to S100β are used for diagnosis and classification of tumors of clear cell origin.

[0005] The discovery of novel calmodulin-related proteins and polynucleotides encoding them has created a need in the art by providing novel compositions that are useful in diagnosing, treating, and preventing cell proliferative disorders and neurological disorders. To satisfy.

[0006] SUMMARY OF THE INVENTION The present invention relates to a novel human calmodulin-related protein (CALREP), C
It is based on the discovery of polynucleotides encoding ALREP and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders and neurological disorders.

[0007] The present invention provides a substantially purified polynucleotide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

[0008] The present invention further provides substantially purified variants having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. The present invention also provides an isolated and purified polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. The present invention also provides an isolated and purified polynucleotide having at least 90% polynucleotide sequence identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. Includes variants.

[0009] The present invention further provides an isolated and purified polypeptide that hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. The present invention provides an isolated and purified polynucleotide that is complementary to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, as well as nucleotides.

[0010] The present invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2 or a fragment of SEQ ID NO: 2, and SEQ ID NO: 2 or SEQ ID NO:
At least 90% of the polynucleotide comprising the polynucleotide sequence of the two fragments
An isolated and purified polynucleotide having the polynucleotide sequence identity of The present invention also provides an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2 or a fragment of SEQ ID NO: 2.

The invention further provides an expression vector comprising at least a fragment of a polynucleotide encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. In another embodiment, the expression vector is contained within a host cell.

The present invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, wherein (a) SEQ ID NO: 1 under conditions suitable for expression of the polypeptide Culturing a host cell comprising an expression vector comprising at least a fragment of a polynucleotide comprising the amino acid sequence of the fragment of SEQ ID NO: 1 or SEQ ID NO: 1; and (b) culturing the polypeptide from the medium of the host cell. And a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1.

[0013] The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 together with a suitable pharmaceutical carrier.

The invention further includes a purified antibody that binds to a polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, as well as a purified agonist and a purified antagonist of said polypeptide. Is included.

The present invention is a method of treating or preventing a cell proliferative disorder associated with reduced CALREP expression, comprising administering to a patient in need of such treatment SEQ ID NO: 1 or SEQ ID NO: 1
A method for treating or preventing a cell proliferation disorder associated with reduced CALREP expression, comprising the step of administering an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of the fragment of .

The present invention also provides a method for treating or preventing a cell proliferative disorder associated with increased expression of CALREP, which comprises administering to a patient in need of such treatment SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. Provided is a method for treating or preventing a cell proliferation disorder associated with an increase in the expression of CALREP, comprising a step of administering an effective amount of an antagonist of a polypeptide having an amino acid sequence.

[0017] The present invention also relates to a method for treating or preventing a neurological disease associated with reduced expression of CALREP, which comprises administering SEQ ID NO: 1 or SEQ ID NO: 1 to a patient in need of such treatment.
Provided is a method for treating or preventing a neurological disease associated with reduced CALREP expression, comprising administering an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of the fragment of

The present invention also provides a method for treating or preventing a neurological disease associated with an increase in the expression of CALREP, which comprises administering to a patient in need of such treatment SEQ ID NO: 1 or SEQ ID NO: 1
A method for treating or preventing a neurological disease associated with an increase in the expression of CALREP, comprising a step of administering an effective amount of an agonist of a polypeptide having the amino acid sequence of the fragment of

The present invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 in a biological sample containing a nucleic acid, the method comprising: Hybridizing a molecule complementary to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 with at least one of the nucleic acids of the biological sample to form a hybridization complex Forming and (b) detecting the hybridization complex, wherein the presence of the hybridization complex determines the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 in the biological sample. Having a correlation with the presence of a polynucleotide encoding a polypeptide comprising And a method for detecting a polynucleotide. In one embodiment, the nucleic acid of the biological sample is amplified by polymerase chain reaction before the step of performing the hybridization.

[0020] (Embodiment of the invention) protein of the present invention, nucleotide sequences, and before describing how this invention is not limited to the particular methodology disclosed herein, protocols, cell lines, limited vectors, and reagents It should be understood that they can be varied in many ways. Further, the terminology used in the present specification is used for the purpose of describing specific examples only, and is intended to limit the scope of the present invention which is limited only by the description of the claims. It should be understood that it is not.

In the detailed description or drawings of the present specification and the appended claims, the terms “a” and “an”, which refer to a singular number, mean that they are not in the context. It should be noted that, other than, there are multiple meanings. Thus, for example, reference to "a certain host cell" includes a plurality of such host cells, and "a certain antibody" includes one or more types of antibodies and well-known to those skilled in the art. And its equivalents.

All technical and scientific terms used herein are, unless otherwise defined,
It has the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All publications mentioned herein are cited for the purpose of describing and disclosing cell lines, vectors, and methodologies reported in the literature that may be used in the context of the present invention. Book. In addition, any disclosure of this specification should not be construed as an admission that such disclosure in the present invention does not precede the prior art.

( Definition ) As used herein, “CALREP” is derived from any species, specifically mammals such as cows, sheep, pigs, mice, horses, and preferably humans, and 1 is a substantially purified CALREP amino acid sequence obtained from any source, synthetic, semi-synthetic, or recombinant.

As used herein, the term “agonist” refers to CA when bound to CALREP
LREP is a molecule that enhances the effect of LREP and prolongs the duration of the effect. Agonists can include proteins, nucleic acids, carbohydrates, and any other molecules that bind to CALREP and modulate its effects.

As used herein, “allelic variant” is an allelic form of the gene encoding CALREP. An allelic variant results from mutation of at least one position in a nucleic acid sequence, resulting in a mutated mRNA or polypeptide, which may or may not alter the structure or function of the mutated mRNA or polypeptide. Certain natural or recombinant genes may have no, one, or multiple allelic forms. In general, the mutations that result in allelic variants result from natural deletions, additions and substitutions of nucleotides. Each of these types of mutations, alone or simultaneously with other mutations, may occur once or twice in a given sequence.
It can occur more than once.

As used herein, the term “mutant” nucleic acid sequence encoding CALREP refers to a polynucleotide that includes substitution, insertion or deletion of different nucleotide residues, and consequently encodes the same CALREP, or the function of CALREP. And a polynucleotide encoding a polypeptide having at least one specific characteristic. This definition includes polymorphisms in the polynucleotide sequence encoding CALREP that have loci other than the normal chromosomal locus, polymorphisms resulting from improper or unexpected hybridization with allelic variants, and CALREP encoding And polymorphisms that can be easily detected using a specific oligonucleotide probe of the polynucleotide to be detected or are difficult to detect. The encoded protein is similarly "mutated"
CALRE, resulting in a silent mutation and consequently a functionally equivalent CALRE
It may include deletion, insertion, and substitution of the amino acid residue serving as P. Intentional amino acid substitutions can be made based on similarities in residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and also amphipathicity, as long as the biological activity of CALREP is retained. . For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having an uncharged polar head group with near hydrophilicity include leucine. Glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.

As used herein, “amino acid” or “amino acid sequence” is a sequence of an oligopeptide, peptide, polypeptide, or protein, or a fragment thereof, and is a naturally occurring molecule or a synthesized molecule. . In this context, a “fragment”, “immunogenic fragment” or “antigenic fragment” is a fragment of CALREP, preferably about 5 to about 15 amino acids, most preferably about 14 amino acids in length. And retains the biological or immunological activity of CALREP. Here, when "amino acid sequence" refers to the amino acid sequence of a naturally-occurring protein molecule, "amino acid sequence" or similar terms refer to the complete, original form of the amino acid sequence associated with the protein molecule described herein. It is not used to limit the amino acid sequence as it is.

[0028] As used herein, "amplification" refers to the production of additional copies of a nucleic acid sequence, usually performed using the polymerase chain reaction (PCR) method well known to those skilled in the art (eg, Dieffenbach, CW). And GS Dveksler (1995) PCR Primer.a Laborato
ry Manual , Cold Spring Harbor Press, Plainview, NY, pp. 1-5).

As used herein, the term “antagonist” refers to a molecule that, when bound to CALREP, reduces the magnitude of the biological or immunological effect of CALREP or shortens the duration of the effect. It is. As an antagonist, CAL
Examples include proteins, nucleic acids, carbohydrates, antibodies, or other molecules that reduce the effects of REP.

As used herein, the term “antibody” refers to an intact antibody molecule, as well as fragments thereof that can bind antigenic determinants such as, for example, Fab, F (ab ′) ₂ , and Fv fragments. I do. Antibodies that bind CALREP polypeptides can be produced by using the entire polypeptide or a fragment thereof containing the small peptide of interest as the antigen for immunization. The polypeptide or peptide used to immunize an animal (eg, a mouse, rat, or rabbit) can be derived from the translation of RNA or can be chemically synthesized and, if necessary, combined with a carrier protein. Can be combined. Commonly used carriers that chemically bind to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). An animal is immunized with the conjugated peptide.

As used herein, the term “antigenic determinant” is a fragment of a molecule (ie, an epitope) that associates with a particular antibody. When a host animal is immunized with a protein or fragment thereof, various regions of the protein can trigger the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein. Such a region or structure is called an antigenic determinant. The antigenic determinant binds to the original antigen (
The immunogen used to induce the immune response).

As used herein, the term “antisense” is a composition that includes a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand"
Used in the sense of a nucleic acid strand complementary to the "sense" strand. Antisense molecules include peptide nucleic acids, and antisense molecules can be created by methods such as synthesis and transcription. Once introduced into the cell, the complementary nucleotide combines with the natural sequence created by the cell to form a duplex, which inhibits further transcription and translation. The expression "minus (-)" is used in the meaning of the antisense strand, and "plus (+)" is sometimes used in the sense of the sense strand.

As used herein, the term “biologically active” is a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to a natural, recombinant, or synthetic CALREP, or oligopeptide thereof, that elicits a specific immune response in appropriate animals or cells and binds to a specific antibody. Ability.

As used herein, the term “complementary” or “complementarity” refers to the spontaneous binding of polynucleotides by forming base pairs under acceptable salt concentrations and temperature conditions. For example, the sequence "AGT" binds to the complementary sequence "TCA". The complementarity between two single-stranded molecules is "partial", in which only some nucleic acids bind, or "complete" if there is complete complementarity between the single-stranded molecules. To "complementary to. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on the binding of nucleic acid strands and in the design and use of peptide nucleic acid (PNA) molecules.

As used herein, “composition containing a predetermined polynucleotide sequence” or “composition containing a predetermined amino acid sequence” refers to any substance containing a predetermined polynucleotide or amino acid sequence. The composition may be in the form of a powder formulation, an aqueous solution, or a sterile composition. Compositions comprising a polynucleotide sequence encoding CALREP or a fragment of CALREP can be used as a hybridization probe. The probe can be stored in a lyophilized state and can be conjugated to a stabilizing agent such as a carbohydrate. In hybridization, the probe can be dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS), and other substances (eg, Denhardt's solution, milk powder, salmon sperm DNA, etc.).

As used herein, a “consensus sequence” is a sequence that is resequenced to separate unneeded bases, and that the nucleotide sequence is determined in the 5 ′ and / or 3 ′ direction using XL-PCR ^™ (Perkin Elmer, Norwalk, Conn.). A computer program for combining the extended and resequenced nucleic acid sequences or fragments, eg, GELVIEW ^™ Fragment Assembly
This is a nucleic acid sequence derived by combining overlapping sequences of two or more types of Incyte clones using a stem (GCG, Madison WI). Consensus sequences may be created by both extension and combination.

As used herein, the expression “correlated with the expression of a polynucleotide” means that the presence of a nucleic acid that is the same as or closely related to a nucleic acid encoding CALREP is detected by Northern analysis. Indicates the presence of mRNA encoding CALREP in the sample, and thus correlates with the expression of transcripts from the gene encoding CALREP.

As used herein, a “deletion” is a change in the nucleotide or amino acid sequence such that one or more nucleotides or amino acid residues are missing.

As used herein, the term “derivative” is a polynucleotide obtained by chemically modifying a polynucleotide sequence encoding CALREP or a polynucleotide sequence complementary thereto. Such chemical modifications of the polynucleotide sequence include, for example, replacement of hydrogen with an alkyl, acyl, or amino group. A derivative polynucleotide encodes a polypeptide that retains at least one of the biological or immunological functions of the original molecule. A derivative polypeptide retains at least one of the biological or immunological functions of the original polypeptide, and has been modified by glycosylation, PEGylation, or some other process. .

As used herein, the term “similarity” refers to a degree of complementarity. There can be partial similarity or complete similarity. The term "(sequence) identity" is a term for "similarity"
Can be replaced by Partially complementary sequences that at least partially inhibit the same sequence from hybridizing to the target nucleic acid are referred to as "substantially similar."
Inhibition of the hybridization between the completely complementary sequence and the target sequence is determined by using a hybridization assay (Southern or Northern blot, solution hybridization, etc.) under conditions of reduced stringency. You can find out. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a sequence or probe that is completely similar (identical) to the target sequence under conditions of low stringency. This does not mean that low stringency conditions allow for non-specific binding. This is because low stringency conditions require that the binding of the two sequences to each other be a specific (ie, selective) interaction. The absence of non-specific binding can be determined by using a second target sequence that has no partial degree of complementarity (ie, less than about 30% similarity or identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

The term “percent identity” or “% identity” is the percentage of sequence similarity when comparing two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, for example, by using the MegAlign program (DNASTAR, Inc., Madison WI). The MegAlign ^™ program uses different methods, such as the Clustal method (eg, Higgins, DG and PM Sharp (1988) Gene 73).
: 237-244) to produce an alignment of two or more sequences.
In the Clustal algorithm, a sequence group is divided into clusters (populations) by checking the distance between both sequences for all pairs of sequences. This cluster group is aligned for each pair, and then the group is aligned. The percent similarity between two amino acid sequences, for example, sequence A and sequence B, is (length of sequence A-number of gap residues in sequence A-number of gap residues in sequence B) / (sequence A and sequence B Total number of residue matches between B) × 100. Cases where the similarity difference between the two amino acid sequences is low or not are not included in the percent similarity calculation. The percent identity between nucleic acid sequences can also be determined by other well-known methods, such as the Jotun Hein method (eg, Hein J. (1990) Methods Enzymol. 183: 626-645).
) Can be counted or calculated. Identity between sequences can also be determined by other well-known methods, for example, by changing hybridization conditions.

“Human artificial chromosomes” (HACs) may comprise DNA sequences of about 6 kb to 10 Mb in size, and are linear miniatures containing all elements necessary for stable division and maintenance of split chromosomes Chromosomes (eg, Harrington, JJ et al. (1997) Nat Ge
net. 15: 345-355).

As used herein, the term “humanized antibody” is an antibody molecule in which the amino acid sequence of the non-antigen binding region has been modified so as to be closer to a human antibody while retaining its original binding ability.

As used herein, the term “hybridization (hybridization)” is the process by which a nucleic acid strand binds to a complementary strand through base pairing.

As used herein, the term “hybridization complex” refers to the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases.
A complex formed by two nucleic acid sequences. The hybridization complex is
Either it is formed in solution (for example, in the case of C ₀ t or R ₀ t analysis), or the nucleic acid is comprised of one nucleic acid present in solution and one solid support (eg, a cell, paper on which the cells or the nucleic acid are immobilized, a membrane, etc.). , A filter, a pin, or another nucleic acid immobilized on a glass slide or other suitable substrate).

As used herein, the term “insertion” or “addition” refers to a nucleotide sequence or amino acid sequence that adds one or more nucleotides or amino acid residues, respectively, as compared to a naturally occurring molecule. Refers to the change.

An “immune response” can refer to a condition associated with inflammation, trauma, an immune disease, or an infectious or genetic disease. These conditions can be characterized by the production of various factors that activate cells and the whole body's defense system, such as cytokines, chemokines, and other signaling molecules.

As used herein, the term “microarray” is an array of individual polynucleotides on a substrate. Substrates include, for example, paper, nylon or other types of membranes, filter paper, chips, glass slides, or any other suitable solid support.

The term “element” as used herein in connection with a microarray
Alternatively, an “array element” is a hybridizable polynucleotide arranged on the surface of a substrate.

As used herein, the term “modulation” refers to altering the activity of CALREP. For example, the modulation may result in an increase or decrease in the activity of the protein, a change in the binding properties, or a change in the biological, functional, or immunological properties of other CALREPs.

As used herein, “nucleic acid” or “nucleic acid sequence” refers to an oligonucleotide, a nucleotide, a polynucleotide, or a fragment thereof, is single-stranded or double-stranded, and is a sense strand or an antisense strand. Refers to some genomic or synthetic DNA or RNA, or peptide nucleic acid (PNA), or DNA-like or RNA-like material. In this context, a “fragment” refers to some functional characteristic of a full-length polypeptide when translated, eg, a polypeptide that retains antigenicity, or a characteristic of a structural domain, eg, an ATP binding site Means a nucleic acid sequence that produces a polypeptide that carries

As used herein, the term “functionally related” or “operably linked” refers to a functionally related nucleic acid sequence. A promoter is operably associated with or linked to a coding sequence if the promoter regulates the transcription of the encoded polypeptide. Although functionally related or operably linked nucleic acid sequences can be in close reading frame, certain genomic sequences, such as repressor genes, are not in close proximity to the encoded polypeptide, but still It binds to an operator sequence that regulates the expression of the polypeptide.

As used herein, the term “oligonucleotide” is a nucleic acid sequence that can be used in a PCR amplification or hybridization assay, or microarray, and is about 6 or more nucleotides in length, up to about 60 nucleotides, preferably Refers to those that are 15 to 30 nucleotides, more preferably 20 to 25 nucleotides. As used herein, the term "oligonucleotide" is substantially synonymous with the terms "ambrimer", "primer", "oligomer", and "probe" as generally defined in the art.

As used herein, “peptide nucleic acid” (PNA) refers to an antisense molecule, ie, an antigenic agent, comprising an oligonucleotide 5 or more nucleotides in length attached to the peptide backbone of amino acid residues having a lysine at the end. I do. Terminal lysine confers solubility to this material. PNAs can preferentially bind to complementary single-stranded DNA or RNA, stop transcript elongation, and extend their lifespan in cells by converting PNA to polyethylene glycol (eg, Nielsen, PE
(1993) Anticancer Drug Des. 8: 53-63).

The term “sample” is used herein in its broadest sense. Biological samples suspected of containing a CALREP-encoding nucleic acid or fragment thereof or CALREP itself include body fluids, extracts from chromosomes, organelles, or cell membranes isolated from cells, cells, ( Genomic DNA, RNA or cDNA (in solution or bound to a solid support), tissue or tissue print (
tissue print) and others.

As used herein, the term “specific binding” or “specifically binds” refers to the interaction of a protein or peptide with agonists, antibodies, and antagonists. This interaction depends on the presence of a particular structure (ie, antigenic determinant, or epitope) on the protein that is recognized by the binding molecule. For example, if the antibody is specific for epitope "A", then in a reaction involving labeled "A" and the antibody, if a protein containing epitope A (ie, unbound, unlabeled A) is present. , The amount of labeled A to which the antibody has bound is reduced.

As used herein, the term “stringent (stringent) conditions” refers to conditions that permit hybridization between a polynucleotide sequence and a claimed polynucleotide sequence. Stringent conditions can be determined, for example, by the concentration of the salt or organic solvent (eg, formamide), the temperature, and other known conditions. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or increasing the hybridization temperature.

As used herein, the term “substantially purified” refers to a substance that has been removed from its natural environment and isolated or separated so that it has more than 60% of its other constituents naturally associated with it. Preferably, it is a nucleic acid or amino acid sequence from which 75% or more, most preferably 90% or more, has been removed.

As used herein, “substitution” refers to replacing one or more nucleotides or amino acids with different nucleotides or amino acids, respectively.

As used herein, “transformation” refers to a process by which foreign DNA enters and changes a recipient cell. This process can occur under natural or artificial conditions using various well-known methods. Transformation can be performed by known methods for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the type of host cell to be transformed and includes, but is not limited to, methods of infecting viruses, electroporation, heat shock, lipofection, and microprojectile bombardment. There may be a method used. The term "transformed" cell refers to the DNA inserted therein.
Is a plasmid that replicates autonomously or a stably transformed cell that can replicate as part of the host chromosome. Also, some of these cells undergo transient expression of the inserted DNA or RNA for a limited time.

As used herein, a “variant” of CALREP is an amino acid sequence in which one or more amino acids have been changed. The variant may contain "conservative" changes, in which the amino acid replaced has similar structural and chemical properties, for example, when leucine is replaced by isoleucine. In rare cases, a variant may change "non-conservatively," in which case, for example, glycine is replaced with tryptophan. Similar minor changes include amino acid deletions or insertions, or both. Substituted without compromising the biological or immunological activity, insertion, or that either deleted amino acids that can cause the deletion, it can be determined using, for example, LASERGENE ^TM software known computer programs such as it can.

( Invention ) The present invention relates to a novel human calmodulin-related protein (CALREP), C
Based on the discovery of polynucleotides encoding ALREP and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders, and neurological disorders.

The nucleic acid encoding CALREP of the present invention can be prepared using a brain tumor cDNA library (BR) using a computer search for amino acid sequence alignment such as BLAST.
AITUT03) was first identified in Insights clone number 2109176 originating from AITUT03). The consensus sequence SEQ ID NO: 2 is the following duplicated and / or extended nucleic acid sequence: Incyte Clone No. 2109176 (origin BRAITUT03), 2653689
(Origin of THYMNOT04), 2878785 (origin of UTRSTUT05), and 959509 (origin of BRSTTUT03).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 shown in FIGS. 1A, 1B, 1C, and 1D. CALREP is 38
It has a length of 6 amino acids, and has the 62nd S (hereinafter referred to as “S62”), T71, S116, S196, S223, T296, S320,
And S369 have eight casein kinase II phosphorylatable sites, T71;
T97, S116, T296, and S320 have five protein kinase C phosphorylatable sites. In addition, the results of both the BLOCKS analysis and the PFAM analysis revealed an EF hand motif generally within the region N324 to L352. Positions 1, 3, 5, 7, and 9 of the EF hand consensus motif are CALREP
D333, E335, S337, Y339, and D341.
The conserved glycine at the sixth position of the EF hand consensus motif is also
Present at G338 in CALREP. Also, from the result of the secondary structure analysis, it is estimated that the putative EF hand adjacent region forms an α helix. As shown in FIG. 2, the EF hand region of CALREP has chemical and structural similarities to the region containing the third EF hand of NB-1 (GI 189081; SEQ ID NO: 3). Note in FIG. 2 that only the relevant EF hand regions of CALREP and NB-1 are shown, and that the amino acid residues are numbered based on their position in the entire protein sequence in the Sequence Listing. . CALREP and NB-
1 share 30% sequence identity within the region shown. In addition, CAL
The phosphorylatable sites in S196 and S369 of REP are conserved in NB-1. The region of unique sequence at about amino acids 21-30 of CALREP is encoded by a fragment of about nucleotides 149-178 of SEQ ID NO: 2. The results of Northern analysis indicate the expression of this sequence in various libraries. At least 67% of such libraries are related to cancerous or proliferative tissue and at least 39% are related to immune response. In addition, 22% of the libraries expressing CALREP are derived from reproductive tissue, and 22% are derived from neural tissue, specifically Alzheimer's disease and tumor-associated brain tissue.

The present invention also encompasses CALREP variants. Preferred CALREP variants have at least one of the functional or structural features of CALREP,
At least about 80%, more preferably at least about 90%
%, Most preferably at least about 95% amino acid sequence identity.

The present invention also includes a polynucleotide encoding CALREP. In certain embodiments, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 2, which encodes CALREP.

The invention also includes variants of the polynucleotide sequence encoding CALREP. Specifically, such a variant polynucleotide sequence is a CALREP
Has at least about 80%, more preferably at least about 90%, and most preferably at least about 95%, polynucleotide sequence identity with a polynucleotide sequence that encodes. Certain embodiments of the present invention provide that SEQ ID NO: 2 has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity with the sequence of SEQ ID NO: 2. Of the present invention. Any of the above described polynucleotide variants may encode an amino acid sequence having at least one of the functional or structural characteristics of CALREP.

As will be appreciated by those skilled in the art, the degeneracy of the genetic code results in a variety of CALREPs, including those that have minimal similarity to the nucleotide sequence of any known naturally occurring gene. Can be created. Thus, the invention includes within its scope all possible nucleic acid sequence variations that are created by selecting combinations based on the possible codon choices. These combinations are made based on the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring CALREP, and all such variations are considered to be specifically disclosed herein. I want to be.

The nucleotide sequence encoding CALREP and variants thereof are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring sequence under conditions of appropriately selected stringency, but have substantially different codons. CAL possessed
It may be beneficial to create a nucleotide sequence that encodes REP or a derivative thereof. In selecting codons, it can be chosen to increase the incidence of peptide expression in a particular prokaryotic or eukaryotic expression host, depending on the frequency with which particular codons are used by the host. Other reasons for substantially altering the nucleotide sequence encoding CALREP and its derivatives so that the encoded amino acid sequence remains unchanged, such as longer half-lives than transcripts made from naturally occurring sequences Creating RNA transcripts with properties.

The scope of the present invention also includes the production of a DNA sequence encoding CALREP or a derivative thereof, or a fragment thereof, by completely synthetic chemistry. Once made, the synthetic gene can be inserted into a variety of available expression vectors and cell lines using well-known reagents. In addition, synthetic chemistry can be used to introduce mutations into the sequence encoding CALREP or any fragment thereof.

Also included within the scope of the present invention are hybridizing polynucleotide sequences according to the claims under various stringency conditions, in particular the polynucleotide sequences of SEQ ID NO: 2 and fragments of SEQ ID NO: 2. There are polynucleotide sequences that can be used (eg, Wahl, GM and SLBerger (1987) Methods Enzymol. 152: 399-407;
And Kimmel, AR (1987) Methods in Enzymol. 152: 507-511). For example,
Stringent salt concentrations are usually less than about 750 mM NaCl and 75 mM.
Less than trisodium acetate, preferably less than about 500 mM NaCl and 75 mM
Less than trisodium acetate, most preferably less than about 250 mM NaCl and less than 25 mM NaCl.
Trisodium acetate less than mM. Hybridization at low levels of stringent conditions is obtained in the absence of an organic solvent, such as formamide, and hybridization at high levels of stringent conditions is at least about 35% formamide, most preferably about 50%. % Formamide. Stringent temperature conditions are usually at least about 30
° C, more preferably at least about 37 ° C, most preferably at least about 42 ° C. It is also known to those skilled in the art to vary other parameters, such as the hybridization time, the concentration of a detergent, such as sodium dodecyl sulfate (SDS), and the presence or absence of carrier DNA. Various levels of stringency,
This can be achieved by combining the above various conditions as necessary. In a preferred embodiment, 750 mM NaCl, 75 mM trisodium acetate, and 1
Hybridization occurs at 30 ° C. in% SDS. In a more preferred embodiment, 500 mM NaCl, 50 mM trisodium acetate, 1% S
DS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (s
Hybridization occurs at 37 ° C. in sDNA). In the most preferred embodiment, 250 mM NaCl, 25 mM trisodium acetate, 1% S
42 in DS, 50% formamide, and 200 μg / ml ssDNA
At C, hybridization occurs. Modifications of these conditions that are beneficial will be apparent to those skilled in the art.

The stringency can also be changed during the washing step performed after hybridization. Stringency conditions for washing can be determined by salt concentration and by temperature. As mentioned above, stringency in the washing process can be increased by lowering the salt concentration or by increasing the temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30 mM NaCl and less than 3 mM trisodium acetate, most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium acetate. Stringent temperature conditions for the washing process are usually at least about 25
° C, more preferably at least about 42 ° C, most preferably at least about 68 ° C. In a preferred embodiment, the washing step is performed at 42 ° C. in 30 mM NaCl, 3 mM trisodium acetate, and 0.1% SDS. In a most preferred embodiment, the washing step comprises 15 mM NaCl, 1.5 mM trisodium acetate,
And in 0.1% SDS at 68 ° C. Other changes to these terms,
It will be clear to those skilled in the art.

Methods for DNA sequencing are well known and commonly available to those of skill in the art, and can be used to practice any of the embodiments of the present invention. In this method, for example, Sequenase which is a Klenow fragment of DNA polymerase I (US Biochemical
Corp. Cleveland OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago IL), or a calibration exonuclease such as that found in the ELONGASE Amplification System released by Gibco BRL (Gaithersburg MD). Enzymes such as those in combination with recombinant polymerases can be used. This process is based on the Hamilton Micro Lab2200 (Hamilton, Reno
, NV), Peltier Thermal Cycler (PTC200; MJ Reserch, Watertown MA) and
It is preferred to automate using equipment such as ABI Catalyst and ABI377 and 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequence encoding CALREP is extended, utilizing the partial nucleotide sequence, using various known PCR-based methods to detect upstream sequences such as promoters and regulatory elements. be able to. For example, in the restriction site PCR method, which is one of the methods that can be used, an unknown sequence is obtained from genomic DNA in a cloning vector using universal primers and nested primers (for example, Sarkar, G. (1993) PCR Methods Applic. 2: 318-322). In another method, the inverse PCR method, an unknown sequence is amplified from a circular template using primers extending in different directions. This template is derived from a restriction fragment containing the known genomic locus and sequences around it. (Eg Triglia, T
Et al. (1988) Nucleic Acids Res 16: 8186). The third method, Capture P
In the CR method, DNA adjacent to a known sequence in human and yeast artificial chromosome DNA is used.
Fragments are amplified by PCR (eg, Lagerstrom, M. et al. (1991) PCR Methods
Applic 1: 111-119). In this method, the recombinant double-stranded sequence can also be incorporated into an unknown fragment of the DNA molecule by digestion and ligation with a plurality of restriction enzymes before performing PCR. Other methods that can be used to fish unknown sequences are also known (eg, Parker, JD et al. (1991) Nucleic
Acids Res 19: 3055-3060). In addition, PCR, nested primers, PromoterFi
Walking in genomic DNA can be performed using the nder ^™ library (Clonte
ch, Palo Alto CA). This process is useful for finding intron / exon junctions without having to screen the library. All PCR
For the method based on the primers, the primers were used in the OLIGO 4.06 Primer Analysis software.
Using commercially available software such as tware (National Biosciences Inc., Plymouth MN) or other suitable programs, at a temperature of 22-30 nucleotides in length, a GC content of 50% or more, and about 68-72 ° C. It can be designed to anneal to the target sequence.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random primed libraries often contain the 5 'region of the gene, and are particularly preferred when oligo d (T) libraries do not yield full-length cDNAs. Genomic libraries can also be useful for extending sequence to a non-transcribed 5 'regulatory region.

A commercially available capillary electrophoresis system can be used to size-analyze the nucleotide sequence of a product of sequencing or PCR and confirm its presence. In particular, capillary sequencing uses a flowable polymer for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) activated by a laser, and is emitted using a CCD camera. The detected wavelength is detected. Output / light intensity is determined by appropriate software (eg, Genotyper ^{™ from} Perkin Elmer).
And Sequence Navigator ^™ ), and the whole process from loading the sample to computer analysis and electronic data display is computer controlled.
Capillary electrophoresis is a method for DN that is present in small amounts in a particular sample.
Particularly suitable for sequencing of small pieces of A.

In another embodiment of the present invention, expression of CALREP, a fragment thereof or a functional equivalent in a suitable host cell by incorporating a polynucleotide sequence encoding CALREP or a fragment thereof into a recombinant DNA molecule. Can be induced.
Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences can also be created, and these sequences can be used for expression of CALREP.

[0078] The nucleotide sequences of the present invention can be recombined using well known methods to modify the sequence encoding CALREP for various purposes. The purpose of this sequence modification includes, but is not limited to, for example, cloning, processing and / or altering the expression of the gene product. DNA from random fragments
Rearrangement and reconstitution of gene fragments and synthetic oligonucleotides by PCR
mbly) allows the nucleotide sequence to be recombined. For example, oligonucleotide-mediated site-directed mutagenesis can achieve the insertion of new restriction sites, altered glycosylation patterns, altered codon preferences, creation of splice variants, introduction of mutations, and the like.

In another embodiment of the present invention, known chemical methods (eg, Caruthers. MH et al. (198)
0) Nucl. Acids Res. Symp. Ser. 7: 215-223; Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232) can be used to synthesize the entire CALREP-encoding sequence or a portion thereof. Alternatively, the protein itself can be produced using chemical methods to synthesize the CALREP amino acid sequence or a fragment thereof. For example, various solid phase techniques (eg, Roberge, JY et al. (1995) Scie
269: 202-204), and automation of the synthesis can be achieved, for example, by using an ABI 431A peptide synthesizer (Perkin Elmer). Further, altering the amino acid sequence of CALREP, and any portion thereof, upon direct synthesis and / or combining with another protein or any portion thereof sequence to produce a mutant polypeptide. Can be.

This peptide can be substantially purified by preparative high performance liquid chromatography (eg, Chiez, RM and FZ Regnier (1990) Methods Enzymol.
182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (eg, Creighton T. (1983) Protei
ns Structure and Molecular Principles , WH Freeman and Co., New York, NY
reference).

In order to express a biologically active CALREP, a nucleotide sequence encoding CALREP or a derivative thereof is regulated by transcription and translation of a coding sequence inserted into a suitable expression vector, ie, a suitable host. Into a vector containing the necessary sequence. These sequences include, for example, in vectors and C
Includes enhancers, constitutive and inducible promoters, and regulatory sequences such as 5 'and 3' terminal untranslated regions in the polynucleotide sequence encoding ALREP. The strength and specificity of the action of such elements can vary. Specific initiation signals are also required for more efficient translation of the sequence encoding CALREP. Such signals include the ATG initiation codon and adjacent sequences, such as the Kozak sequence. If CALREP and its initiation codon and upstream control sequences are inserted into the appropriate expression vector, no other transcriptional or translational control signals are required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal including the ATG initiation codon must be provided. In addition, the start codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be from a variety of sources, both natural and synthetic. Inclusion of appropriate enhancers in the particular cell line used can increase the efficiency of expression (eg, Scharf, D. et al. (1994) Results
Probl. Cell Differ. 20: 125-162).

[0082] To prepare an expression vector containing a sequence encoding CALREP and appropriate transcriptional and translational control regions, methods well known to those skilled in the art can be used. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques (eg, Sambrook, J. et al. (1989) Molecular Cloning, AL ).
aboratory Manual , Cold Spring Harbor Press, Planview NY, ch. 4, 8 and Aus
ubel, FM, etc. (1995, and periodic supplements) Current Protocol in Molecular Biolo
gy , John Wiley & Sons, New York, NY, ch. 9, 13, and 16).

A variety of expression vector / host systems can be utilized for retaining and expressing CALREP encoding sequences. These include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector; yeast transformed with a yeast expression vector; virus expression Insect cell lines infected with vectors (eg baculovirus); viral expression vectors (eg cauliflower mosaic virus (CaMV))
Plant cell lines transformed with a bacterial tobacco mosaic virus (TMV) or bacterial expression vector (eg, Ti or pBR322 plasmid); or animal cell lines. The present invention is not limited by the host cell used.

In bacterial systems, a variety of cloning and expression vectors may be selected depending upon the use of the polynucleotide encoding CALREP. For example, CALRE
Routine cloning, subcloning, and propagation of a polynucleotide encoding P can be accomplished using a multifunctional E. coli vector such as, for example, Bluescript (Stratagene) or pSport1 ^™ plasmid (GIBCO BRL). The incorporation of sequences encoding CALREP into the various cloning sites of the vector to disrupt the lacZ gene allows colorimetric screening to identify transformed bacteria containing the recombinant molecule.
In addition, these vectors are used for in vitro transcription, dideoxy sequencing,
It may be useful for single strand rescue by helper phage, and for the generation of nested deletions in cloned sequences (eg, Van
Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509). For example, when a large amount of CALREP is required for antibody production, a vector that induces high-level expression of CALREP can be used. For example, vectors containing a strong inducible T5 or T7 bacteriophage promoter can be used.

For the production of CALREP, a yeast expression system can also be used. For example α
Factor, a variety of vectors containing constitutive or inducible promoters such as alcohol oxidase, and PGH, yeast Saccharomyces cerevisiae (Saccharo
myces cerevisiae ) and Pichia pastoris ( Pichia pastoris)
). Furthermore, such vectors induce either the extracellular secretion or retention of the expressed protein in cells, allowing the integration of foreign sequences into the host genome for stable expression. (Eg Ausubel,
Supra; and Grant et al. (1987) Methods Enzymol. 153: 516-54; Scorer, CA et al. (1994)
Bio / Technology 12: 181-184).

[0086] Plant systems can also be used for expression of CALREP. For example, CaMV 35S
And a viral promoter, such as the 19S promoter, alone or TMV (
(1987) EMBO J 6: 307-311) can be used to enhance transcription of the sequence encoding CALREP. Or RUBI
Plant promoters such as the small subunit of SCO and heat shock promoters may be used (eg, Coruzzi, G. et al. (1984) EMBO J 3: 1671-1680; Broglie, R.
(1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Ce.
ll Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or transfection with a pathogen (eg, Ho
bbs, S. or Murry, LE McGraw Hill Yearbook of Science and Technology (1
992) McGraw Hill NY, pp 191-196).

For mammalian cells, a variety of viral-based expression systems may be utilized. When an adenovirus is used as an expression vector, a sequence encoding CALREP is replaced with a late promoter and a tripartite lead sequence.
r sequence) can be linked to the adenovirus transcription / translation complex. Insertion into a non-essential E1 or E3 region of the viral genome results in an infectious virus expressing CALREP in host cells (eg, Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81: 3655-3659).
In addition, to increase expression in mammalian host cells, Rous sarcoma virus (RS
V) A transcription enhancer such as an enhancer can be used.

The use of human artificial chromosomes (HACs) can also provide fragments of DNA larger than those that can be integrated into and expressed from a plasmid. For therapeutic purposes, 6 kb to 10 Mb HACs can be constructed and supplied using conventional delivery methods (liposomes, polycationic amino polymers, or vesicles).

To ensure long-term recombinant protein production in a mammalian system, stable expression of CALREP in a cell line is desirable. For example, using an expression vector that can contain the viral origin of replication and / or the endogenous expression element and the selectable marker gene on the same vector or on separate vectors,
Sequences encoding REP can be transformed into cell lines. After the introduction of the vector, the cells are allowed to grow for approximately 1-2 days in a concentrated medium, and then are switched to a selective medium. The purpose of the selectable marker is to confer resistance to the selectable drug and allow cells that, based on their presence, definitely express the introduced sequence to grow and be recovered. Resistant clones of stably transformed cells can be grown using tissue culture techniques appropriate for the cell type.

Any number of selection systems can be used to recover transformed cell lines. Selection systems include, but are not limited to, herpes simplex virus thymidine kinase gene (tk) and adenine phosphoribosyltransferase gene (tk).
apr) and used in tk ^- or apr ^- cells, respectively (eg Wigler
(1977) Cell 11: 223-232 and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to the aminoglycosides neomycin and G-418, als or pat confers resistance to chlorsulfuron, phosphinotricin acetyltransferase. (Eg, Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570, Colberre-Ga
rapin, F. et al. (1981) J. Mol. Biol. 150: 1-14, and Murry (supra)). Alternative genes that can be used for selection include, for example, trpB and hisD, which alter substances required by cells for metabolism (eg, Hartman, SC and RC Mull).
igan (1988) Proc. Natl. Acad. Sci. 85: 8047-8051). Visible markers such as anthocyanins, green fluorescent protein (GFT)
, Β-glucuronidase and its substrate, β-D-glucuronoside, or luciferase and its substrate, luciferin. These markers can be incorporated not only to identify transformants, but also to quantify the amount of transient or stable protein expression in a particular expression vector system (eg, Rhodes, CA et al. (1995) Methods Mol. Biol. 55: 121-131).

The presence / absence of marker gene expression also indicates the presence of the gene of interest, but its presence and expression may need to be confirmed. For example, CALREP
Is inserted into the marker gene sequence, transformed cells into which the sequence encoding CALREP has been incorporated can be identified based on the lack of function of the marker gene. Alternatively, the marker gene can be placed in tandem with the sequence encoding CALREP, and both can be under the control of a single promoter. Expression of the marker gene in response to induction, ie, selection, usually simultaneously represents expression of the tandemly arranged sequences.

In general, host cells that contain a nucleic acid sequence encoding CALREP and that express CALREP can be identified by various methods well known to those of skill in the art. Such methods include, but are not limited to, DNA-DNA or DNA-RN.
A-hybridization, amplification by PCR, and protein bioassays or immunoassays, including techniques using membranes, solutions, or chips to detect and / or quantify nucleic acids and proteins, and the like.

[0093] Immunological methods for detecting and measuring CALREP expression using either polyclonal or monoclonal antibodies specific for this protein are well known. Such methods include, but are not limited to, enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter (FACS), and the like. A two-site, monoclonal-based immunoassay utilizing a monoclonal antibody reactive to two non-interfering epitopes on the CALREP polypeptide is preferred, but a competitive binding assay may be used. Good. These and other assays are well known (eg, Hampton, R. et al. (1990) Serological Methods, a Laboratory ).
Manual , APS Press, St. Paul MN; Coligan, JE et al. (1997 and regularly published supplements) Current Protocols in Immunology, Greene Pub. Associates and Wile
y-Interscience, New York, NY; and Maddox, DE et al. (1983) J. Exp. Med. 158:
1211-1216).

Various labeling and conjugation techniques are well known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing a labeled hybridization probe / PCR probe for detecting a sequence closely related to the polynucleotide encoding CALREP include oligo labeling, nick translation, end labeling, and labeling. PCR amplification using the modified nucleotides. Alternatively, the sequence encoding CALREP or any fragment thereof
It may be cloned into a vector for the production of NA probes. Such vectors are well known and commercially available and can be used, for example, in T7, T3, or SP6.
RNA probes can be synthesized in vitro by adding a suitable RNA polymerase and labeled nucleotides such as These methods are
Various commercially available kits, such as Pharmacia Upjohn (Kalamazoo, MI); Promega (Ma
dison WI); and those provided by US Biochemical Corp. (Cleveland OH). Suitable reporter molecules, or labels, that can be used to facilitate detection include radionuclides, enzymes, fluorescers, chemiluminescent or dye agents, substrates, cofactors, inhibitors, magnetic And the like.

[0095] Host cells transformed with a nucleotide sequence encoding CALREP can be cultured under conditions suitable for expressing the protein in the cell culture medium and recovering it therefrom. Proteins produced by the transformed cells are secreted or retained intracellularly, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing polynucleotides encoding CALREP can be used to transfer CALRE through the cell membrane of prokaryotic or eukaryotic cells.
It can be designed to include a signal sequence that induces P secretion.

In addition, host cell strains can be chosen for their ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cuts off the "prepro" portion of the protein can also be used to identify protein targeting, folding, and / or action. A variety of host cells (eg, CHO, HeLa, MDCK, 293, and WI38) that have specific cellular machinery and characteristic mechanisms for post-translational action are available from American
It is available from the Type Culture Collection (ATCC; Bethesda, MD) and can be selected from among them to ensure correct modification and processing of the foreign protein to be introduced.

In another embodiment of the present invention, the natural, modified or recombinant nucleic acid sequence encoding CALREP is ligated to a heterologous sequence to translate the fusion protein in any of the host systems described above. Can be caused. For example, a chimeric CALREP protein containing a heterologous molecule that can be recognized by commercially available antibodies can facilitate the selection of inhibitors of CALREP activity from a peptide library. Such molecules include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, And hemagglutinin (HA
). GST, MBP, Trx, CBP, and 6-His allow purification of immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and their cognate fusion proteins on metal chelating resins, respectively. Become. FLAG, c-myc, and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize their epitope tags. By recombining the fusion protein to include a proteolytic cleavage site between the sequence encoding CALREP and the heterologous protein sequence, CALR
The EP can be separated from the heterologous molecule after purification. Methods for expression and purification of fusion proteins are described in Ausubel, FM et al. (1995 and periodic supplements).
) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N
Y, ch10. Various commercially available kits may be used to rapidly perform expression and purification of the fusion protein.

In yet another embodiment of the present invention, radiolabeled CALREP can be synthesized in vitro using the TNT ^™ rabbit reticulocyte lysate or wheat germ extract system (Promega, Madison, WI). . These systems are T7, T3, or SP
6 links transcription and translation of protein coding sequences functionally related to the promoter. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵ S-methionine.

The production of CALREP fragments can be carried out not only by recombinant production but also by direct peptide synthesis using solid-phase technology (eg, Creighto).
n, supra, pp. 55-60). Protein synthesis can be performed manually or automatically. Automation of synthesis can be achieved, for example, using an Applied Biosystems 431A peptide synthesizer (The Perkin-Elmer Corp., Norwalk, Conn.). Various fragments of CALREP can be synthesized separately and ligated to create a full-length molecule.

( Treatment ) CALREP and EF hand domain, specifically EF of human-derived NB-1
There are chemical and structural similarities between the hand domain (GI 189081). In addition, CALREP is expressed in proliferating and neural tissues. Therefore, CALREP is thought to play a role in cell proliferation disorders and neurological diseases.

Thus, in certain embodiments, CALREP or a fragment or derivative thereof may be administered to a patient for the treatment or prevention of a cell proliferative disorder associated with reduced CALREP expression. Such diseases include, but are not limited to, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal Nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers such as adenocarcinoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically
Adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, Cancers such as testicular, thymic, thyroid, and uterine cancers; and immune disorders such as leukemia, lymphoma, rheumatoid arthritis, dermatitis, inflammation, systemic lupus erythematosus, and other autoimmune disorders, and the like. .

In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof is administered to a patient for the treatment or prevention of a cell proliferative disorder, including but not limited to those listed above. obtain.

In another embodiment, a medicament comprising substantially purified CALREP together with a suitable pharmaceutical carrier for the treatment or prevention of a cell proliferative disorder, including but not limited to those listed above. The composition can be administered to a patient.

In yet another embodiment, an agonist that modulates the activity of CALREP may be administered to a patient for the treatment or prevention of a cell proliferative disorder, including but not limited to those listed above.

In yet another embodiment, an antagonist of CALREP may be administered to a patient for the treatment or prevention of a cell proliferative disorder associated with increased CALREP expression. Such diseases include, but are not limited to, those listed above. In some embodiments, using an antibody that specifically binds to CALREP directly as an antagonist, or indirectly using the antibody as a targeting or delivery mechanism to deliver a drug to cells or tissues in which CALREP is expressed Can be.

In another embodiment, a vector expressing a molecule complementary to a polynucleotide encoding CALREP is used for the treatment or prevention of a cell proliferative disorder, including but not limited to those listed above. It can be administered to a patient.

In another embodiment, CALREP or a fragment or derivative thereof may be administered to a patient for the treatment or prevention of a neurological disorder associated with reduced CALREP expression. Such diseases include, but are not limited to, restlessness, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasm, cerebral infarction, dementia, depression Disease, diabetic neuropathy, Down syndrome, tardive dyskinesia, dystonia, epilepsy, head trauma, Huntington's chorea, peripheral nerve disease,
Examples include multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia, and Tourette's syndrome group.

In another embodiment, a vector capable of expressing CALREP or a fragment or derivative thereof may be administered to a patient for the treatment or prevention of a neurological disorder, including but not limited to those listed above. .

In another embodiment, a pharmaceutical composition comprising substantially purified CALREP together with a suitable pharmaceutical carrier for the treatment or prevention of a neurological disease, including but not limited to those listed above. Can be administered to the patient.

In yet another embodiment, an agonist that modulates the activity of CALREP may be administered to a patient for the treatment or prevention of a neurological disorder, including but not limited to those listed above.

In yet another embodiment, an antagonist of CALREP may be administered to a patient for the treatment or prevention of a neurological disorder associated with increased CALREP expression. Such diseases include, but are not limited to, those listed above. In some embodiments, using an antibody that specifically binds to CALREP directly as an antagonist, or indirectly using the antibody as a targeting or delivery mechanism to deliver a drug to cells or tissues in which CALREP is expressed Can be.

In another embodiment, a vector expressing a molecule complementary to a polynucleotide encoding CALREP is administered to a patient for the treatment or prevention of a neurological disorder, including but not limited to those listed above. May be administered.

In another embodiment, a protein, antagonist, antibody, agonist,
Either the complementary sequence, or the vector, can be administered in combination with other suitable agents. One of skill in the art would be able to select an appropriate drug for use in combination therapy based on conventional pharmaceutical principles. By combining therapeutic agents, a synergistic effect that is effective in treating or preventing the various diseases described above can be obtained. By using this method, the therapeutic effect can be increased with lower doses of each drug, and the possibility of causing side effects can be reduced.

[0114] CALREP antagonists can be prepared using well-known methods. In particular, purified CALREP can be used to generate antibodies or to screen libraries of drugs to identify those that specifically bind to CALREP. Antibodies to CALREP can also be generated using known methods. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments made from Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use.

A variety of hosts, such as goats, rabbits, rats, mice, etc., can be immunized by injecting CALREP or fragments or oligopeptides thereof with immunological properties to generate antibodies. Various adjuvants, depending on the host species, can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, Whole limpet hemocyanin and dinitrophenol. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

The oligopeptide, peptide, or fragment thereof used to elicit antibodies to CALREP preferably has 5 or more amino acids, more preferably 10 amino acids.
It has an amino acid sequence consisting of at least two amino acids. Also, these sequences are preferably identical to a portion of the amino acid sequence of the original protein, and include the entire amino acid sequence of the small, naturally occurring molecule. A short stretch of CALREP amino acids
It can be fused with stretches of other proteins such as keyhole limpet hemocyanin (KLH) to produce antibodies against the chimeric molecule.

[0117] Monoclonal antibodies to CALREP are produced in unrestricted proliferating cell lines (continuum) in culture.
(a continuous cell line) using a technique for producing antibody molecules. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, EBV-hybridoma technology, and the like (eg, Kohl
er, G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) J. Immunol. Metho
ds 81: 31-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; C
ole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, “chimeric antibodies”, such as techniques for splicing mouse antibody genes to human antibody genes, to obtain molecules with appropriate antigen specificity and biological activity.
Techniques developed for the production of (eg, Morrison, SL
Natl. Acad. Sci. 81: 6851-6855; Neuberger, MS et al. (1984).
Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, CALRE may be applied in a known manner, applying well-known techniques for the production of single-chain antibodies.
Single-chain antibodies specific for P can be created. Antibodies with related specificities but different idiotypic configurations can be generated by chain shuffling from random combinatorial immunoglobulin libraries (
For example, see Burton DR (1991) Proc. Natl. Acad. Sci. 88: 11120-3).

In addition, literature (eg, Orlandi, R. et al. (1989), Proc. Natl. Acad. Sci. 86: 383)
3-3837; by screening a panel of highly specific binding reagents or a recombinant immunoglobulin library, as disclosed in Winter, G. et al. (1991) Nature 349: 293-299). Alternatively, antibodies can be produced by inducing production in a lymphocyte population in vivo.

[0120] Antibody fragments which contain specific binding sites for CALREP can also be generated.
Such fragments include, but are not limited to, F (ab ') ₂ fragments and F (ab') fragments that can be generated by digestion of antibody molecules with pepsin.
) Fab fragments that can be created by reducing the disulfide bridges between the _two fragments. Alternatively, a Fab expression library may be generated so that monoclonal Fab fragments with the desired specificity can be identified quickly and easily (eg, Huse, WD et al. (1989) Science 256: 1275-128).
1).

[0121] A variety of immunoassays can be utilized for screening to identify antibodies with the desired specificity. Various protocols for competitive binding assays or radioimmunoassays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. In such an immunoassay, the amount of a complex formed between CALREP and its specific antibody is measured. Two-site monoclonal based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes
mmunoassay) is preferred, but a competitive binding assay may be used (Maddox, supra).

In another embodiment of the present invention, a polynucleotide encoding CALREP, or any fragment or complement thereof, can be used for therapeutic purposes. In some embodiments, in situations where it is desirable to inhibit the transcription of mRNA, CA
A complementary sequence to a polynucleotide encoding LREP can be used. Specifically, cells can be transformed with a sequence complementary to the polynucleotide encoding CALREP. Thus, using complementary molecules or fragments, CA
It can modulate LREP activity, that is, regulate gene function. Such techniques are now well known, and sense or antisense oligonucleotides, or larger fragments, can be designed from various locations in the coding and regulatory regions of the CALREP coding sequence.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or expression vectors derived from various bacterial plasmids, are used for delivery of nucleotide sequences to target organs, tissues, or cells. be able to. A vector expressing a nucleic acid sequence complementary to the polynucleotide of the gene encoding CALREP can be prepared using methods well known to those skilled in the art (
See, for example, Sambrook et al. (Supra) and Ausubel et al. (Supra).

The gene encoding CALREP can be disrupted by transforming cells or tissues with an expression vector that expresses CALREP-encoding polynucleotide or a fragment thereof at high levels. Using such a product,
Non-translatable sense or antisense sequences can be introduced into cells. Such vectors, even when not incorporated into DNA, continue to transcribe the RNA molecule until the vector is destroyed by an endogenous nuclease. Such transient expression can last for more than a month, even for non-replicating vectors, and even longer if appropriate replication elements are part of the vector system.

As described above, a sequence complementary to the control region, 5 ′ region, or regulatory region (signal sequence, promoter, enhancer, and intron) of the gene encoding CALREP, that is, an antisense molecule (DNA, RNA or By designing PNA), the way of gene expression can be changed. Oligonucleotides derived from the transcription initiation site, e.g., the region approximately between +10 and -10 of the start site, are preferred. Similarly, inhibition can be achieved using triple helix base pairing. Triple helix pairing is useful because it inhibits the ability of the double helix to unwind sufficiently for binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triple helical DNA have been described in the literature (eg, Huber, BE and BI Carr, Molecular and Immunologic Ap.).
proaches , Futura Publishing Co, Mt Kisco NY, see Gee, JE et al. (1994)). Complementary sequences, ie, antisense molecules, can also be designed to prevent transcript binding to ribosomes and inhibit mRNA transcription.

[0126] Ribozymes, which are enzymatic RNA molecules, can also be used to catalyze the specific cleavage of RNA. In the mechanism of ribozyme action, a ribozyme molecule hybridizes to a complementary target RNA in a sequence-specific manner, followed by endonucleolytic cleavage. Examples of ribozymes that can be used include:
There are artificially synthesized hammerhead ribozyme molecules that can specifically and effectively catalyze the cleavage of CALREP-encoding sequences by endonucleases.

[0127] Specific ribozyme cleavage sites in the RNA that can be targeted are identified by first scanning the ribozyme cleavage sites containing the sequences GUA, GUU and GUC in the target molecule. Once identified, it is possible to evaluate the characteristics of the secondary structure that stops the function of the oligonucleotide for a short RNA sequence consisting of 15 to 20 ribonucleotides corresponding to the region of the target gene including the cleavage site. . The suitability of a candidate target is also determined by the ribonuclease protection assay (ribonucl
This can be assessed by measuring the accessibility for hybridization with complementary oligonucleotides by an ease protection assay.

[0128] Complementary ribonucleic acid molecules and ribozymes of the invention can be made by well-known methods for the synthesis of nucleic acid molecules. These techniques include oligonucleotide chemical synthesis techniques such as solid phase phosphoramidite chemical synthesis. Or,
RNA molecules can be created by in vivo and in vitro transcription of a DNA sequence encoding CALREP. Such a DNA sequence may be T7 or SP
6 can be incorporated into various vectors having a suitable RNA polymerase promoter. Alternatively, a cDNA construct that synthesizes constitutive RNA can be introduced into a cell line, cell or tissue.

An RNA molecule can be modified to increase its stability in a cell or to increase its half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' ends of the molecule, and phosphorothionate (rather than phosphodiesterase linkages) within the backbone of the molecule. phosph
orothioate) or 2'O-methyl. This method (
concept) is originally used in the production of PNAs and can be extended to all of these molecules as follows. That is, inosine, queosine, as well as by modification of acetyl-, methyl-, thio-, and similar forms of adenine, guanine, cytidine, thymine, and uridine that are not readily recognized by endogenous endonucleases. This scheme can be extended to all of these molecules by including previously rarely used bases such as, and wybutosine.

A number of methods are available for introducing vectors into cells or tissues,
The methods are equally suitable for in vivo , in vitro and ex vivo uses. In the case of ex vivo treatment, there is a method of introducing a vector into stem cells collected from a patient, growing the clone as a clone for autologous transplantation, and returning the same patient. Delivery by transfection, liposome injection or delivery by polycation amino polymer (Goldman, CK et al. (1997) Nature Biotechnology 1
5: 462-66; incorporated herein by reference) can be performed using methods well known in the art.

[0131] Any of the above treatments can be applied to any suitable subject, including, for example, dogs, cats, cows, horses, rabbits, monkeys, and most preferably mammals, such as humans.

In yet another embodiment of the present invention, the pharmaceutical composition is administered with a pharmaceutically acceptable carrier to achieve the therapeutic effect described above. Such a pharmaceutical composition may be a CAL
It may consist of REP, antibodies to CALREP, CALREP mimetics, agonists, antagonists or inhibitors. The pharmaceutical compositions are administered alone or together with one or more other drugs, such as, for example, stabilizers, using a sterile, biocompatible pharmaceutical carrier. Such carriers include, but are not limited to, saline, buffered saline, glucose or water. Such compositions can be administered to the patient alone or in combination with other drugs or hormones.

[0133] The administration route of the pharmaceutical composition used in the present invention is not limited to the following, but may be oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, Examples include intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration, local administration, sublingual administration, and rectal administration.

These pharmaceutical compositions, in addition to the principal component, are suitable pharmaceutically-acceptable agents, such as excipients and additives, which facilitate the processing of the active ingredient into pharmaceutically usable formulations. Carrier. For more information on dispensing or dosing techniques, see Remington's Phar
It can be found in the latest edition of maceutical Sciences (Maack Publishing Co, Easton PA).

Pharmaceutical compositions for oral administration can be formulated in suitable dosage forms using pharmaceutically acceptable carriers well known in the art. With such a carrier, the pharmaceutical composition can be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for patients to take. it can.

Pharmaceutical preparations for oral administration are prepared by combining the main component and solid excipients, pulverizing the resulting mixture as required, and preparing tablets or dragee cores.
) Is obtained by processing a mixture of granules (after grinding, if desired). If necessary, suitable additives can be added. Suitable additives include sugar or protein fillers such as sugars including lactose, sucrose, mannitol or sorbitol, starches such as corn, wheat, rice, potatoes, methylcellulose, hydroxypropylmethylcellulose or carboxyl. There are celluloses such as sodium methylcellulose, gums such as gum arabic or tragacanth, and proteins such as gelatin or collagen. If desired, disintegrating or solubilizing agents may be added, or the cross-linked polyvinyl pyrrolidone, agar, alginic acid such as sodium alginate, or a salt thereof such as sodium alginate.

Dragee cores are used by coating appropriate tablets with a concentrated sugar solution or the like. Solutions for making the tablets include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or tablets for tablet identification or to indicate the amount or dosage of the principal component.

Orally administrable formulations include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a tablet, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with excipients or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the principal component is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycol, with or without stabilizers.

Pharmaceutical preparations for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, and synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aminopolymers of non-lipid polycations can also be used for delivery. Appropriate agents or stabilizers may be added to the suspension, as desired, to increase solubility and allow for the preparation of highly concentrated solutions.

For topical or nasal mucosal administration, preparations are made with an appropriate penetrant for the particular barrier to be permeated. Such penetrants are well-known in the art.

The pharmaceutical compositions of the present invention may be prepared by any of the methods well known in the art, including, for example, conventional mixing, dissolving, granulating,
Sugar coating, levigating, emulsifying, encapsulating, entrapping,
Alternatively, it is produced by freeze-drying.

The pharmaceutical composition can be provided as a salt and can be formed with various acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In addition, preferred formulations include those in the range of 4.5-5.5, buffered before use, from 1 mM to 5 mM.
There are lyophilized powders that contain all or any of 0 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol.

After the pharmaceutical composition has been prepared, it can be placed in an appropriate container and labeled further for use in treating the indicated conditions. For the administration of CALREP, such a label indicates the amount, frequency, and manner of administration.

Pharmaceutical compositions suitable for use in the present invention are those that contain the active ingredient in an effective amount to achieve the desired purpose. Determination of an effective amount can be made well within the ability of those skilled in the art.

For any compound, the therapeutically effective amount can be estimated initially from assays of neoplastic cells or cell media, usually in any animal model such as mouse, rabbit, dog, pig. This animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine effective doses and routes for administration in humans.

A therapeutically effective amount is an amount of an active ingredient that ameliorates a symptom or condition, for example, CALREP or a fragment thereof, a CALREP antibody, a CALREP agonist, antagonist, or inhibitor. Therapeutic efficacy and toxicity of such compounds is determined by standard pharmaceutical procedures in cell culture media or using laboratory animals.
For example, ED50 (therapeutically effective amount in 50% of the population, 50% effective amount) and L
It can be determined as D50 (50% lethal dose of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that achieve the ED50 with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, as well as the route of administration.

The correct dosage will be chosen by the attending physician in view of factors related to the patient in need of treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient and to maintain the desired effect. Factors to consider include the severity of the disease state, the patient's general health, the patient's age, weight, and gender, diet, time and frequency of administration, concomitant medications, response sensitivity, and resistance / response to treatment. And the like. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the clearance rate of the particular formulation.

Typical doses are in the range of 0.1-100,000 μg, with a total dose of up to about 1 g, depending on the route of administration. Guidance as to particular dosages or delivery methods can be found in the literature commonly available to practitioners in the art. Those skilled in the art will employ different dosage forms for nucleotides than for proteins and inhibitors. Similarly, the mode of delivery of a polynucleotide or polypeptide will depend on the particular cell, condition, location, and the like.

Diagnosis In another embodiment, antibodies that specifically bind to CALREP may be used to diagnose a disease characterized by CALREP expression, or to be treated with CALREP or a CALREP agonist, antagonist, or inhibitor. Can be used in assays for monitoring patients who have Antibodies useful for diagnosis can be made in the same manner as for therapeutics described above. CALREP
CALR in human body fluids, cell or tissue extracts
There is a method using an antibody or a label to detect EP. The antibody may be used with or without modification, and may be labeled by binding to the reporter molecule either covalently or non-covalently. Some are described above, but various reporter molecules are known and can be used.

For example, ELISA (enzyme linked immunoassay), RIA (radioimmunoassay)
As well as various protocols for measuring CALREP, such as FACS (Fluorescent Display Cell Sorter), are well known in the art, and provide the basis for diagnosing alterations or abnormalities in CALREP expression. Normal value of CALREP expression,
That is, the standard value is established by binding a body fluid or a cell extract obtained from a normal subject, preferably a mammal, to a CALREP antibody under conditions suitable for complex formation. The standard complex formation can be quantified by various methods, preferably by using photometric means. The amount of CALREP expressed in affected tissue samples and control samples of the subject's biopsy tissue is compared to a standard value. A parameter for diagnosing the disease is established from the deviation between the standard value and the value of the subject.

In another embodiment of the invention, a polynucleotide encoding CALREP may be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. This polynucleotide is used to detect and quantify gene expression in biopsy tissue where CALREP expression may be related to disease. The diagnostic assay is CA
It can be used to distinguish whether LREP is present, absent, or overexpressed, or to monitor regulation of CALREP levels in therapeutic intervention.

In one embodiment, identifying a nucleic acid sequence encoding CALREP using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequences, encoding CALREP or closely related molecules. Can be. The specificity of the probe, ie, whether the probe is in a very highly specific region (eg, 10 unique nucleotides in the 5 ′ regulatory region) or a region of low specificity (eg, especially the 3 ′ coding region). Source, and the stringency of hybridization or amplification (high, medium or low), whether the probe identifies only naturally occurring CALREPs, or identifies allelic or closely related sequences Is determined.

Probes can also be used to detect closely related sequences, preferably
At least 50% nucleotides based on any sequence encoding CALREP
Should be included. The hybridization probe of the present invention comprises DNA or R
NA and may be derived from the sequence of SEQ ID NO: 2 or from a genomic sequence that includes introns, promoters and enhancer elements of the naturally occurring CALREP gene.

As a means for preparing a hybridization probe specific to DNA encoding CALREP, a method of cloning a nucleic acid sequence encoding CALREP or a CALREP derivative into a vector for producing an mRNA probe is used. is there. Such vectors are well known and commercially available and can be used for in vitro RNA probe synthesis by adding an appropriate RNA polymerase or an appropriate labeled nucleotide. Hybridization probes can be labeled with various reporter molecules. For example, it can be labeled with a radionuclide such as ³² P or ³⁵ S with an enzyme label such as alkaline phosphatase which binds to the probe via an avidin / biotin binding system.

[0155] Polynucleotide sequences encoding CALREP can be used for diagnosis of a disease associated with CALREP expression. Examples of such diseases include:
Without limitation, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polymorphism Blood, psoriasis,
Primary thrombocythemia; cancers such as adenocarcinoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, gastrointestinal tract Cancers such as duct, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterus cancer; and leukemia, lymphoma, rheumatism Cell proliferative disorders such as osteoarthritis, dermatitis, inflammation, systemic lupus erythematosus, and immune disorders such as other autoimmune disorders; and restlessness, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder , Catatonia, brain neoplasm, cerebral infarction, dementia, depression, diabetic neuropathy, Down syndrome, tardive dyskinesia, dystonia, epilepsy, head trauma, Huntington's chorea, peripheral nervous disease, multiple sclerosis, nerve Fibromatosis, pa Mention may be made of neurological disorders such as Kinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia, and Tourette syndrome. CALREP-encoding polynucleotide sequences can be obtained from Southern or Northern blots, dot blots or other membrane-based techniques, PCR techniques, dipstick assays (test strips) using patient tissues or body fluids. Method), pin technology, and ELISA assays, or in microarrays, to detect changes in CALREP expression. Such qualitative or quantitative test methods are well known in the art.

In certain embodiments, nucleotide sequences encoding CALREP may be useful, particularly in assays that detect the presence of related disorders, such as those listed above. The nucleotide sequence encoding CALREP can be labeled by standard techniques and added to a patient's body fluid or tissue sample under conditions suitable for forming hybridization complexes. After an appropriate incubation time, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the biopsy or extracted sample is significantly different from the amount of signal in the comparable control sample, then the nucleotide sequence has hybridized to the nucleotide sequence of the sample and encodes CALREP in the sample. The occurrence of a change in the level of the nucleotide sequence indicates the presence of the associated disease. Such assays can also be used in animal studies, clinical trials, or in monitoring the treatment of an individual patient to assess the effectiveness of a particular therapeutic treatment.

To establish a basis for the diagnosis of diseases associated with the expression of CALREP, a normal, ie standard, expression profile is established. This standard profile is established by combining extracts of bodily fluids or cells from normal subjects, either animals or humans, with CALREP-encoding sequences or fragments thereof under conditions suitable for hybridization or amplification. I can do it. The amount of standard hybridization can be quantified by comparing values obtained in experiments using known amounts of substantially purified CALREP to values obtained in normal subjects. Standard values obtained from a normal sample can be compared to values obtained from a sample of a patient exhibiting symptoms of the disease. The presence of the disease is confirmed using the deviation between the standard value and the subject value.

[0158] Once the disease has been identified and the treatment protocol initiated, hybridization assays are performed periodically to determine whether the level of expression in the patient has begun to approach that observed in normal patients. Can be evaluated. The results from continuous assays can be used to determine the efficacy of treatment over a period of days or months.

For cancer, the presence of abnormal amounts of transcripts in patient biopsies
Indicates a predisposition to the occurrence of the disease. In other words, it can be a means of detecting a disease before actual clinical symptoms appear. This type of definitive diagnosis allows healthcare professionals to take preventive measures or initiate aggressive treatment earlier and prevent the onset and further progression of cancer.

Another diagnostic use of oligonucleotides designed from sequences encoding CALREP may be in PCR. Such oligomers can be chemically synthesized, made using enzymes, or made in vitro. Oligomers, including fragments of the polynucleotide encoding CALREP or polynucleotides complementary to the polynucleotide encoding CALREP, are used under optimal conditions to identify a particular gene or condition. Oligomers can also be used under low stringency conditions for the detection or quantification of closely related DNA or RNA sequences.

Methods that can be used to quantify CALREP expression include the use of radiolabeled or biotin-labeled nucleotides, the use of co-amplification of control nucleic acids, and complementing the experimental results. The use of a standard graph curve drawn or the like (eg, Melby, PC et al. (1993) J. Immunol. Me
thods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). For quantification of large numbers of samples, the oligomers of interest are present in multiple dilutions and can be further quantified by performing ELISA-type assays that can be quickly quantified using a spectrophotometer or colorimetrically. Speed can be increased.

In another embodiment, oligonucleotides or larger fragments from any of the polynucleotide sequences disclosed herein can be used as targets in a microarray. By using microarrays, the expression levels of many genes can be monitored simultaneously (to generate transcript images), and gene variants, mutations, and polymorphisms can be identified. This information can be used to determine gene function, understand the genetic basis of disease, diagnose disease, and develop therapeutics and monitor their activity.

A microarray may be prepared and analyzed using known methods (eg, Bren
nan, TM et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl.
Acad. Sci. 93: 10614-10619; Baldeschweiler et al., (1995) PCT application WO95 / 251116;
Shalon, D et al. (1995) PCT application WO95 / 35505; Heller, RA et al. (1997) Proc. Natl. A
cad. Sci. 94: 2150-2155; see Heller, MJ et al. (1997) U.S. Patent No. 5,605,662).
.

In another embodiment of the invention, nucleic acid sequences encoding CALREP can be used to create useful hybridization probes for mapping naturally occurring genomic sequences. This sequence may map to a particular chromosome, a particular region of the chromosome, or an artificial chromosome construct. Examples of artificial chromosome products include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (B
ACs), bacterial P1 constructs or monosomal cDNA libraries (eg, Price, CM (1993) Blood Rev. 7: 127-134, and Trask, BJ (1991) Tren).
ds Genet. 7: 149-154).

Fluorescence in situ hybridization (FISH) may correlate with other physical chromosome mapping techniques and genetic map data (Meyers, RA (ed.)
Molecular Biology and Biotechnology, VCH Publishers New York, NY, pp. 96
See Heinz-Ulrich et al. (1995) at 5-968). Examples of genetic map data can be found in various scientific journals and in Online Mendelian Inheritance in Man (OMIM). The position of the sequence encoding CALREP on the physical chromosome map and the specific disease (
Or a predisposition to a particular disease)
The area of NA can be determined. The nucleotide sequences of the present invention can be used to detect differences in the gene sequence of normals, carriers, or patients.

Physical mapping techniques, such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers, can be used to enlarge the genetic map. In many cases, even if the number or arm of a particular human chromosome is unknown, the gene arrangement on the chromosome of another mammal, such as a mouse, will reveal the relevant marker. New sequences can be assigned to chromosomal arms or parts thereof by physical mapping. This can provide valuable information to researchers investigating disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been localized to a particular genomic region, for example, as ataxia telangiectasia (AT) has been located at 11q22-23 (see, eg, Gatti, RA et al. (1988) Nature 336: 577-580). Once coarsely located, any sequence that maps to that region could represent a relevant gene for further study, or a regulatory gene. The nucleotide sequences of the present invention can also be used to detect differences in chromosomal location between normals, carriers, and patients, caused by translocations, inversions, and the like.

In another embodiment of the present invention, CALREP, its catalytic or immunogenic fragments, oligopeptides can be used for screening compounds in various drug screening techniques. Fragments used in such screening may be in free form in solution, in a form attached to a solid support, in a form attached to a cell surface, or in a cell. Can be The binding complex formation between CALREP and the drug under test can be measured.

[0168] Alternative drug screening techniques allow for high-throughput screening of compounds with appropriate binding affinity for the protein of interest (
See, for example, Geysen et al. (1984) PCT application WO 84/03564). In this method, CALR
For EP applications, a number of different miniature compounds to be tested are synthesized on a solid substrate such as a plastic pin or other surface. The compound to be tested is
React with LREP or a fragment thereof and wash. The bound CALREP is then detected by methods well known in the art. Also, purified CALREP can be coated directly on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured using a non-neutralizing antibody and immobilized on a solid support.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding CALREP specifically compete with the compound under test for binding to CALREP. In this method, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants of CALREP.

In yet another embodiment, even if the molecular biology technology has not been developed to date, the new technology may have properties known to date for nucleotide sequences (eg,
Any technique based on, but not limited to, the triplet genetic code and specific base pairing) can use a nucleotide sequence encoding CALREP in the technique.

The following examples are merely illustrative of the present invention and are not intended to limit the present invention to these examples.

[0172]

【Example】

1. Preparation of cDNA Library A BRAITUT03 cDNA library was prepared using RNA isolated from brain tumor tissue collected from the left anterior lobe of a 17-year-old white female at the time of resection of a meningeal lesion. Lesions showed grade 4 fibrous giant and small cell astrocytomas. Family history included benign hypertension and cerebrovascular disease.

[0173] The frozen tissue was used as a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann I
nstruments, Inc., Westbury, NJ) and homogenized and dissolved in the guanidinium isothiocyanate solution. RNA was isolated according to Stratagene's RNA isolation protocol (Stratagene, La Jolla, CA). RNA was extracted twice with acidic phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and DNase treated. Poly (A +) RNA is from Qiagen O
It was isolated using a ligotex kit (QIAGEN, Inc., Chatsworth, CA).

Using poly (A +) RNA, cDNA was synthesized and SuperScript ^™ Plasmid Sy
stem for cDNA Synthesis and Plasmid Cloning (Part No. 18248-013; Gibco / BRL, G
aithersburg, MD) according to the recommended protocol. Use cDNA for Sepharose CL4B column (Part No. 275105, Pharmacia Amersh
am Biotech, Piscataway, NJ).
NA was linked to pSport I (Part No. 15382-013, Gibco BRL). Next, this recombinant plasmid was transformed into DH5a ^™ competent cells (Part No. 18258-012, Gibco / BRL).

2 Isolation and sequencing of cDNA clones Plasmid DNA was released from the cells and was
(No. 26173; QIAGEN, Inc.). We used the recommended protocol,
The following points have been changed. (1) Bacteria were cultured in 1 ml of sterile Terrific Broth (Part No. 22711, Gibco / BRL) containing 25 mg / L carbenicillin and 0.4% glycerol. (2) After incubating the culture solution for 19 hours, the cells were dissolved in 0.3 ml of lysis buffer. (3) After isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. This DN
Sample A was stored at 4 ° C.

The sequencing of this cDNA was performed using Peltier Thermal Cyclers (MJ Research, Water
town, MA PTC200) and Applied Biosystems 377 DNA Sequencing System
Using Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with ms
The reading frame was determined by the method of Sanger et al. (1975, J. Mol. Biol. 94: 441f).

3 Similarity Search of cDNA Clones and Their Analogous Proteins Databases such as GenBank, SwissProt, BLOCKS, and Pima II were searched using the nucleotide sequence and / or amino acid sequence of the Sequence Listing as a query sequence. These databases contain annotated sequences already identified, and BLAS
Regions having similarity were searched from this database using T (for Basic Local Alignment Tool) (for example, Altschul. SF (1993) J. Mol. Evol
36: 290-300; see Altschul et al. (1990) J. Mol. Biol. 215: 403-410).

BLAST creates an alignment of both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of the alignment, BLAST is particularly useful in identifying exact matches, ie, homologs of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms can be used to handle primary sequence patterns and gap penalties for secondary structure (see, eg, Smith T et al. (1992) Protein Engineering 5: 35-51). The sequence disclosed herein is at least 49 nucleotides in length, with one unnecessary base.
2% or less (where N is recorded other than A, C, G, or T)
.

The BLAST method searches for matches between a query sequence and a sequence in the database, evaluates the statistical significance of any found sequence match, and reports only matches that meet a user-selected significance threshold. . The threshold in the present application was set to 10 ^{-25 for} nucleotides and 10 ^-8 for peptides.

The nucleotide sequence of Incyte was searched in the GenBank database of primates (pri), rodents (rod), and other mammalian sequences (mam), and the amino acid sequence deduced from the same clone was then retrieved. , GenBank's Functional Protein Database, mammals (mamp), vertebrates (vrtp), and eukaryotes (eukp).

In addition, sequences identified from the cDNA library can be analyzed using an appropriate analysis program, for example, BLOCKS, to identify gene sequences encoding conserved protein motifs. BLOCKS is available in the PROSITE database (Bairoch, A.
(1997) Nucleic Acids Res. 25: 217-221), a weighted matrix analysis algorithm based on short amino acid segments or blocks. The BLOCKS algorithm helps classify genes with unknown functions (Heni
koff S. and Henikoff GJ, Nucleic Acids Research (1991) 19: 6565-6572).
Blocks of 3 to 60 amino acids in length correspond to the most highly conserved regions of the protein. The BLOCKS algorithm compares the query sequence with the block's weighted scoring matrix in the BLOCKS database. BL
Blocks in the OCKS database are calibrated against protein sequences of known function from the SWISS-PROT database to determine the probability distribution of matches. Similar databases, such as the protein fingerprint database PRINTS, can also be searched using the BLOCKS algorithm (Artwood, TK et al.).
(1997) J. Chem. Inf. Comput. Sci. 37: 417-424.). PRINTS is, for example, SwissP
Based on non-redundant sequences obtained from sources such as rot, GenBank, PIR, and NRL-3D.

[0182] The BLOCKS algorithm uses a query sequence between the BLOCKS or PRINTS database.
Search for matches and evaluate the statistical significance of the matches found. BLOCKS or PRINTS
Search matches are evaluated at two levels: local similarity and overall similarity
be able to. The degree of local similarity is measured by a score, and the overall similarity
Is measured by a score ranking and a probability value. Highest rankin
A score of 1000 or more for a BLOCKS match for a group
When the matched block was calibrated against SWISS-PROT, the false positive level was 0.5%.
Indicates that it is within the centile. Similarly, if the probability value is 1.0 × 10 ^-3 Less than means that a match occurs more than once per 1000 searches
Is shown. Cut-off score of 1000 or more and 1.0 × 10^-3Less cut-off
Only matches with probability value are functional analysis of protein sequence in sequence listing
Be considered.

The nucleic acid and amino acid sequences in the Sequence Listing can also be analyzed using PFAM. PFAM
Is a Hidden Markov Model (HMM) based protocol useful in the search for protein families. HMM is a probabilistic method for analyzing the consensus primary structure of a gene family (eg, Eddy, SR (1996) Cur. Opin. Str. Biol.
6: 361-365.).

The PFAM database searches for well-characterized protein domains using two high quality alignment routines, seed alignment and full alignment (eg, Sonnhammer, ELL et al. 19
97) Proteins 28: 405-420.). Species alignment utilizes the hmmls program, a program that searches for local matches, and a non-redundant set of PFAM databases. The whole alignment makes use of the hmmfs program, a program that searches for multiple fragments in long sequences, such as repetitive sequences and motifs, and all sequences in the PFAM database. Results That score 100 "bits", the sequence may mean that the likelihood to see the true match to the model or comparison sequences is two ^hundred times or more. When using the SWISS-PROT sequence as a model or comparison sequence, cut-off scores ranging from 10 to 50 bits are usually used for individual protein families.

Two other algorithms, SIGPEPT and TM, both based on the HMM algorithm described above (see, eg, Eddy, supra and Sonnhammer), identify potential signal sequence and transmembrane domains, respectively. SIGPEPT is SWISS-PROT
Produced using a protein sequence with a signal sequence annotation derived from It contains about 1413 non-redundant signal sequences, 14 to 36 amino acid residues in length. TMs were generated using transmembrane domain annotation as well. It contains approximately 453 non-redundant transmembrane sequences, including 1579 transmembrane domain segments. An appropriate HMM model is created using the above-described array and has a high correlation coefficient,
That is, the accuracy was increased using a known SWISS-PROT signal peptide sequence or transmembrane domain sequence until a measurement of the correctness of the analysis was obtained. SWISS-PR as test set
Using the protein sequence from the OT database, the 11-bit cutoff score as determined above correlates with 91-94% of true positives and about 4.1% of false positives, The correlation coefficient is about 0.87 to 0.90. An 11-bit score for TM generally gives the following results. That is, 75% true positives and 1.72% false positives, and a correlation coefficient of 0.76. Each search evaluates the statistical significance of the matches found, and only matches with a score of at least 11 bits are reported.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of a gene transcript, which involves labeling a nucleotide sequence with the RN of a particular cell type or tissue.
Hybridization with the membrane to which A is bound (for example, Samb
rook, supra, ch. 7; and Ausubel, FM et al., supra, ch. 4 and 16.).

Using similar computer techniques applied to BLAST, search for identical or closely related molecules in nucleotide databases such as the GenBank or LIFESEQ ^™ database (Incyte). This analysis takes much less time than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be varied to determine whether a particular match is classified as an exact match or similar.

The reference value of the search is a product score, which is defined by the following equation. (Sequence match (%) × Maximum BLAST score (%)) / 100 This product score takes into account both the degree of similarity between two sequences and the match in sequence length. For example, if the product score is 40, the match is accurate within an error of 1-2%, and if the score is 70, the match is exact. Similar molecules are usually identified by selecting those that show a product score of 15-40, while those with lower scores are identified as closely related molecules.

The results of the Northern analysis are reported as a list of libraries in which transcripts encoding CALREP are generated. Abundance and percent abundance are also reported. Abundance directly reflects the number of times a particular transcript is present in the cDNA library, and percent abundance is the abundance divided by the total number of sequences detected in the cDNA library.

5 Extension of Sequence Encoding CALREP The nucleic acid sequence of Insight No. 2109176 was used to design an oligonucleotide primer to extend a partial nucleotide sequence to full length.
One primer was synthesized to initiate extension in the antisense direction and the other primer was synthesized to extend the sequence in the sense direction. Using these primers, the known CALREP sequence was extended "outside" to generate an amplicon containing a new, unknown nucleotide sequence of the region of interest. The first primer was designed using OLIGO ^™ 4.06 (National Biosciences, Plymouth, MN), or other suitable program, and was about 22 to about 30 nucleotides in length, 50% or more. It was designed to have a GC content and anneal to the target sequence at a temperature of about 68 to about 72 ° C. Stretches of any nucleotides that would result in hairpin structure and primer-primer dimerization were excluded.

This sequence was extended using a selected human cDNA library (Gibco / BRL). If more than one step extension is necessary or desirable, an additional set of primers is designed to further extend the known region.

High fidelity amplification is achieved by thorough mixing of the enzyme with the reaction mixture according to the instructions in the instructions for the XL-PCR ^™ kit (Perkin Elmer). 40 pmol
When starting amplification from each of the primers and all other components of the kit at the recommended concentration, the following parameters were used using a Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA), ie, step 194 ° C. 1 minute (initial denaturation) Step 2 65 ° C for 1 minute Step 3 68 ° C for 6 minutes Step 4 94 ° C for 15 seconds Step 5 65 ° C for 1 minute Step 6 68 ° C for 7 minutes Step 7 Further Steps 4 to 6 15 cycle repetition Step 8 94 ° C. for 15 seconds Step 9 65 ° C. for 1 minute Step 10 68 ° C. for 7 minutes 15 seconds Step 11 Repeat Steps 8 to 12 for 12 cycles Step 12 72 ° C. for 8 minutes Step 134 ° C. (the temperature PCR was carried out.

An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose minigel to determine whether the sequence extension reaction was successful. Were determined. Select the largest product or band, cut out of the gel,
Purification was performed using QIAQuick ^™ (QIAGEN Inc., Chatsworth, Calif.) And the single-stranded nucleotide overhang was cut off using the Klenow enzyme to create blunt ends that facilitate recombination and cloning.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl
Of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added, and the mixture was incubated for 2-3 hours at room temperature or overnight at 16 ° C. Competent E. coli cells (in 40 μl of appropriate culture medium)
The ligation mixture was transformed using μl and cultured in 80 μl SOC medium (eg, Sembrook et al., supra, Appendix A, p. 1). After incubation at 37 ° C. for 1 hour, all transformation mixtures were washed with Luria Bertani containing 2 × Carb (L
B) Placed on agar (eg, Sembrook et al., Supra, Appendix A, p. 2). At a later date,
Several colonies were randomly selected from each plate and the appropriate commercially available sterile 96
150 μl of liquid LB contained in each well of a well microtiter plate
/ 2xCarb medium. Further at a later date, 5 μl of each overnight culture was transferred into a non-sterile 96-well plate, diluted 1:10 with water, and then 5 μl of each sample was transferred to a PCR array.

For PCR amplification, 18 μl of the enriched PCR reaction mixture (3.3 ×) containing 4 units of rTth DNA polymerase, vector primers, and / or primers specific for the gene used in the extension reaction were added to each well. Added. The amplification was carried out under the following conditions: Step 1 at 94 ° C. for 60 seconds Step 2 at 94 ° C. for 20 seconds Step 3 at 55 ° C. for 30 seconds Step 4 at 72 ° C. for 90 seconds Step 5 Steps 2 to 4 were further repeated 29 cycles Step 6 72 Performed at 74 ° C. (maintained) at 180 ° C. for 180 seconds.

An aliquot of the PCR reaction was run on an agarose gel with molecular weight markers. The size of the PCR product was compared to the original partial cDNA, the appropriate clone was selected, ligated to the plasmid and sequenced.

Similarly, using the nucleotide sequence of SEQ ID NO: 2 and 5 ′ using the procedure described above
It is possible to obtain regulatory sequences, obtain oligonucleotides designed for extension in the 5 'direction, and obtain appropriate genomic libraries.

6 Labeling and Use of Hybridization Probe Using a hybridization probe made from the sequence of SEQ ID NO: 2,
Screen cDNA, mRNA and genomic DNA. Particular mention is made of the labeling of oligonucleotides of about 20 base pairs, but generally the same procedure is used for larger cDNA fragments. OLIGO4.06 (National Bioscience
Oligonucleotides were designed using state-of-the-art software such as) and 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN). , B
oston, MA). The labeled oligonucleotide was applied to a Sephadex G-25 ultrafine resin column (Pharmacia & Upjohn, Kalamazoo, MI).
). An aliquot containing per minute 10 ⁷ counts of labeled probe, endonuclease AseI, Bgl II, EcoRI, Pst I, Xba1 or PvuII (DuP
ont NEN, Boston, Mass.) in a general membrane-based hybridization analysis of human genomic DNA digested with one.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH).
Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are washed sequentially at room temperature under stepwise increasing stringency conditions up to 0.1 × aqueous sodium citrate and 0.5% sodium dodecyl sulfate. After exposing the blot to XOMAT AR ^™ film (Kodak, Rochester, NY) for several hours, the hybridization patterns are visually compared.

7 Synthesize oligomers on the surface of the substrate using a microarray chemical coupling procedure and an inkjet device (see, eg, Baldeschweiler et al., Supra). In yet another method, cDNA fragments or oligonucleotides are attached to a substrate using an array similar to that used in dot blotting (slot blotting) using a vacuum system, thermal, UV, mechanical or chemical binding procedures. Placed on the surface of and bonded. Typical arrays can be made by hand or by using commercially available materials and machines and can have an appropriate number of elements. After hybridization, the microarray is washed to remove unhybridized probes, and the level and pattern of fluorescence are determined using a scanner. Scanned images can be analyzed to assess the degree of complementarity and relative abundance of nuclear probes that hybridize to elements on the microarray.

[0201] Full-length cDNAs or expressed sequence tags (ESTs) can comprise elements of a microarray. Fragments suitable for hybridization are, for example, LASERG
The selection can be made using well-known software such as ENE ^™ . Full-length cDNAs or ESTs corresponding to one of the nucleotide sequences of the invention or randomly selected from a cDNA library related to the invention are placed on a suitable substrate, for example a glass slide. The cDNA is immobilized on a glass slide by, for example, UV crosslinking followed by heat and chemical treatment and drying (eg, Schena. M et al. (1995) Science 270: 467-470; and Shalon, D. et al. (19
96) Genome Res. 6: 639-645). Fluorescent probes are made and used for hybridization to elements on a substrate. This substrate is analyzed according to the procedure described above.

8. Sequence Complementary to the Complementary Polynucleotide CALREP or a sequence complementary to any portion thereof may be used to reduce, ie, inhibit, the expression of naturally occurring CALREP. Although the use of oligonucleotides of about 15 to about 30 base pairs is specifically noted, essentially the same procedure can be used for smaller or larger sequence fragments. The appropriate oligonucleotides are designed using OLIGO ^™ 4.06 software and the coding sequence of CALREP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent promoter binding. To inhibit translation, a complementary oligonucleotide is designed so that the ribosome does not bind to transcripts encoding CALREP.

9 CALREP Expression CALREP expression and purification is achieved using a bacterial or virus-based expression system. For expression of CALREP in bacteria, the cDNA is subcloned into a suitable expression vector containing an inducible promoter or a component resistance gene that drives high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter, lac operator control sequences, and the like. The recombinant vector is transformed into a suitable bacterial host, for example, BL21 (DE3). Isopropyl-1-thio-β-D-galactoside (I
When induced by PTG), constituent-resistant bacteria express CALREP. Expression of CALREP in eukaryotic cells is well known as baculovirus,
This is achieved by infecting insect or mammalian cell lines with the recombinant Autographica californica polynucleosis virus (AcMNPV). The non-essential polyhedrin gene of the baculovirus is replaced with cDNA encoding CALREP by either homologous recombination or bacterial-mediated gene transfer using transfer plasmid-mediated. The virus's infectivity is maintained, and the strong polyhedrin promoter drives high levels of cDNA transcription. The recombinant baculovirus is used to infect Spondoptera frugiperda (Sf9) insect cells, most often human hepatocytes, in most cases. In the latter case, it is necessary to make additional genetic modifications to the baculovirus (Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci.
USA 91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945).

[0204] In most expression systems, for example, FLAG or 6-Hi, which allows for a rapid, one-step affinity purification of recombinant fusion proteins from crude cell lysates
s-like peptide epitope tag or glutathione S transferase (
GST) is used to synthesize the fusion protein. Schistosoma
immobilized glutathione (Pharmacia, P.) under conditions that maintain the activity and antigenicity of the protein by GST, a 26 kilodalton enzyme from Japonicum.
iscataway, NJ). After purification, GST
The molecule is CALREP by proteolysis at the specifically recombined site.
Can be disconnected from A commercially available monoclonal and polyclonal anti-FLAG antibody (Eastman Ko
dak, Rochester, NY). 6-His, a stretch of six consecutive histidine residues, allows the metal chelating resin (Q
IAGEN Inc, Chatworth, CA). Methods for protein expression and purification are described in Ausubel. FM et al. (1995 and regular supplements) Current Protoco
ls in Molecular Biology, John Wiley & Sons, New York, NY, ch 10,16. Purified CALREP obtained by these methods can be used directly in assays for the following activities.

[0205]10 Demonstration of CALREP activity In assays for CALREP activity, Ca²⁺Using the overlay system, C
ALREP Ca²⁺(Weis, K et al. (1994) J. Biol. Chem. 26)
9: 19142-19150). Transfer purified CALREP to nitrocellulose membrane
Transfer and secure. The membrane was buffered (60 mM KCl, 5 mM MgCl _Two , 10 mM imidazole HCl, pH 6.8) three times and 1 μCi of [⁴⁵Ca²⁺] (NEN
-DuPont, Boston, MA) for 10 minutes in this buffer.
You. Unbound [⁴⁵Ca²⁺] Is removed by washing with water and the membrane is dried.
Dry. Bound to the membrane⁴⁵Ca²⁺] By autoradiography
And quantitate using an image analysis system and software. CALREP
Activity was detected on the membrane [⁴⁵Ca²⁺]].

11 Functianl assays CALREP function is assessed by expressing CALREP encoding sequences at physiologically high levels in mammalian cell culture systems. cDNA
Is subcloned into a mammalian expression vector that has a strong promoter that allows it to be expressed at high levels. The selected vector is pCMV SPORT ^TM (
Life Technologies, Gaithersburg, MD) and pCR ^TM 3.1 (Invitrogen, Carlsba
d, CA), and both vectors have a cytomegalovirus promoter. 5-10 μg of the recombinant vector is transiently transfected into human cell lines, preferably from endothelial cells or hematopoietic cells, using either liposome formulations or electroporation. In addition, another plasmid having a sequence encoding the marker protein is co-infected with 1-2 μg. Marker protein expression provides a means to distinguish transfected cells from non-transfected cells and allows reliable prediction of cDNA expression from recombinant vectors. Examples of selectable marker proteins include, for example, Green Fluorescent Pr
otein (GFP) (Clontech, Palo Alto, CA), CD64 or CD64-GFP fusion protein and the like. Identify transfected cells expressing GFP or CD64-GFP,
In order to evaluate its properties, for example, its apoptotic state, flow cytometry (flow cytometry) (FCM), a technique using an automatic laser beam, is used. FCM detects the uptake of fluorescent molecules and diagnoses events that occur before or concurrent with quantitative cell death. Such events include propidium iodide with D
Changes in nuclear DNA content as measured by staining NA, scattered light and 90%
Changes in cell size and granularity as measured by exposure to sideways scattered light, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, cells as measured by reactivity with specific antibodies Changes in surface and intracellular protein expression, and changes in plasma membrane composition as measured by binding of fluorescein-bound alexin V protein to the cell surface. The method of flow cytometry is described in Ormerod, MG (1994) Flow Cytometry , Oxford.
, New York, NY.

The effect of CALREP on gene expression can be assessed using a sequence of CALREP and a highly purified population of cells transfected with either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from non-transfected cells using magnetic beads (DYNAL, Lake Success, NY) coated with either human IgG or antibodies to CD64. Purification of mRNA from cells can be performed using methods well known to those skilled in the art. Expression of mRNA encoding CALREP and other genes of interest can be analyzed by Northern blot analysis or microarray technology.

12 Production of Antibodies Specific for CALREP Polyacrylamide gel electrophoresis (PAGE) (eg, Harrington, MG
(1990) Methods Enzymol. 182: 488-495), or CALREPs substantially purified using other purification techniques are used to immunize rabbits according to standard protocols to produce antibodies.

[0209] Alternatively, the amino acid sequence of CALREP can be obtained by using LASERGENE ^™ software (DNASTAR I
nc.) to determine the region of high immunogenicity, synthesize the corresponding oligopeptide and use it to produce antibodies using methods well known to those skilled in the art. Techniques for selecting appropriate peptide sequences and producing antibodies are well known to those skilled in the art. The selection of suitable epitopes, such as epitopes near the C-terminus or in hydrophilic regions, is described in the literature (see, eg, Ausubel et al., Supra, ch. 11).

In general, oligopeptides having a length of 15 residues were synthesized by chemistry of the fmoc method using a peptide synthesizer Model 431A of Applied Biosystems, and M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS
) By keyhole limpet hemocyanin (KLH, Sigma, S
t. Louis, MO) (see, eg, Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity of the resulting antiserum, for example, the peptide is conjugated to plastic, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further reacted with radioactive iodine-labeled goat anti-rabbit IgG. Let react.

13 Purification of Spontaneous CALREP Using Specific Antibodies Spontaneous CALREP or recombinant CALREP is substantially purified by immunoaffinity chromatography using antibodies specific for CALREP. The immunoaffinity column is a CnBr-activated Sepharose (Pharmacia & Upjoh)
It is constructed by covalently linking the activated chromatography resin as in n) with the CALREP antibody. After binding, the resin is blocked and washed according to the instructions for use.

The culture containing CALREP is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of CALREP (eg, with a high ionic strength buffer in the presence of a detergent). This column is used for antibody and CALREP
Eluted under conditions that break binding (e.g., a buffer of pH 2-3 or a high concentration of chaotropic ions such as urea or thiocyanate ions),
Collect CALREP.

14 Identification of Molecules That Interact with CALREP CALREP or a biologically active fragment thereof is labeled with ¹²⁵ I Bolton Hunter reagent (see, eg, Bolton et al. (1973) Biochem. J. 133: 529). Candidate molecules previously arranged in the wells of a multiwell plate are labeled with CALRE
Incubate with P, wash and assay wells with labeled CALREP complex. Using the data obtained with different concentrations of CALREP, the association, affinity, and number values of the candidate molecule and CALREP are calculated.

Various modifications of the method and system of the invention described herein without departing from the scope and spirit of the invention will be apparent to those skilled in the art. Although the invention has been described with reference to particularly preferred embodiments, it should be understood that the true scope of the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1A: CALREP amino acid sequence (SEQ ID NO: 1) and nucleic acid sequence (SEQ ID NO: 2)
FIG. Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Softw
are Engineering Co., Ltd., San Bruno, CA).

FIG. 1B: CALREP amino acid sequence (SEQ ID NO: 1) and nucleic acid sequence (SEQ ID NO: 2)
FIG. Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Softw
are Engineering Co., Ltd., San Bruno, CA).

FIG. 1C: CALREP amino acid sequence (SEQ ID NO: 1) and nucleic acid sequence (SEQ ID NO: 2)
FIG. Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Softw
are Engineering Co., Ltd., San Bruno, CA).

FIG. 1D: CALREP amino acid sequence (SEQ ID NO: 1) and nucleic acid sequence (SEQ ID NO: 2)
FIG. Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Softw
are Engineering Co., Ltd., San Bruno, CA).

[Fig.2] LASERGENE ^™ software multi-sequence alignment program (DNAST
EF hand domain of CALREP (AR Inc, Madison WI)
FIG. 2 shows an amino acid sequence alignment between 2109176; SEQ ID NO: 1) and human NB-1 (GI 189081; SEQ ID NO: 3).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C12N 1/15 4H045 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21 / 02 C 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12P 21/02 A61K 37/02 C12Q 1/68 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , M , RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Korey, Neil Sea, USA 40940, Mountain View, CA # 30, Dale Avenue 1240 (72) Inventor Gegler, Karl Jay Menlo Park Oakland Aveni, 94025, California, United States View 1048 (72) Inventor Patterson, Chandra 94040, California, USA Mountain View Leland Avenue 2189 F-term (Reference) 4B024 AA01 AA11 CA04 CA06 DA06 EA04 GA11 HA12 HA15 4B063 QA01 QA19 QQ08 QQ43 QR08 QR32 QR42 QR56 QS25 QS34 QX Q02 4B064 AG01 AG26 AG27 CA02 CA10 CA19 CA20 CC24 DA01 DA13 4B065 AA26X AA92X AA93Y CA24 CA44 CA46 4C084 AA01 AA02 AA06 AA07 AA17 BA01 BA22 CA53 DB59 DC50 ZA012 ZC032 4H045 AA10 FA30 EA30 FAA

Claims (22)

[Claims] 1. A substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. 2. A substantially purified variant having at least 90% amino acid sequence identity to the sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide of claim 3. 5. An isolated and purified polynucleotide that hybridizes under stringent conditions with the polynucleotide of claim 3. 6. An isolated and purified polynucleotide that is complementary to the polynucleotide of claim 3. 7. An isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2 or a fragment of SEQ ID NO: 2. 8. An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide of claim 7. 9. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 7. 10. An expression vector comprising at least a fragment of the polynucleotide of claim 3. 11. A host cell comprising the expression vector according to claim 10. 12. A method for producing a polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, wherein (a) the host of claim 11 under conditions suitable for expression of the polypeptide. A method for producing a polypeptide comprising the sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, comprising: culturing cells; and (b) recovering the polypeptide from the medium of the host cell. 13. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable pharmaceutical carrier. 14. A purified antibody that specifically binds to the polypeptide of claim 1. 15. A purified agonist of the polypeptide of claim 1. 16. A purified antagonist of the polypeptide of claim 1. 17. A method for treating or preventing a cell proliferation disorder, comprising administering an effective amount of the pharmaceutical composition of claim 13 to a patient in need of such treatment. Prevention methods. 18. A method for treating or preventing a cell proliferation disorder, comprising administering an effective amount of the antagonist of claim 16 to a patient in need of such treatment. . 19. A method for treating or preventing a neurological disease, comprising a step of administering an effective amount of the pharmaceutical composition of claim 13 to a patient in need of such treatment. . 20. A method for treating or preventing a neurological disease, comprising a step of administering an effective amount of the antagonist of claim 16 to a patient in need of such treatment. 21. A method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in a biological sample containing a nucleic acid, comprising: (a) the polynucleotide of claim 6; Hybridizing at least one of the nucleic acid materials of the biological sample to form a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex is determined. Encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 in a biological sample comprising nucleic acids, the process correlating with the presence of a polynucleotide encoding the polypeptide in the biological sample. A method for detecting a polynucleotide that does. 22. The method according to claim 21, wherein the nucleic acid of the biological sample is amplified by polymerase chain reaction before the step of performing the hybridization.